HEPES-involved hydrothermal synthesis of Fe₃O₄ nanoparticles and their biological application

Abstract

A simple one-step hydrothermal route was developed for the synthesis of magnetite (Fe₃O₄) nanoparticles in HEPES solution, using FeCl₂ as the Fe(II) precursor. It was found that HEPES played an important role in the fabrication of Fe₃O₄ nanoparticles, where HEPES could act as a weak antioxidant to prevent the complete oxidation of Fe(II) to Fe(III). The possible formation mechanism of HEPES-coated Fe₃O₄ nanomaterials was also discussed. In the reaction system, the influence of inorganic anions on the morphologies of Fe₃O₄ nanostructures was also investigated. The as-synthesized spherical Fe₃O₄ nanoparticles with a concentration of 5–100 μg mL⁻¹ exhibited nontoxicity towards HUVEC normal cells, indicating their potential application in biology. Combining the preparation of Ag and Au nanoparticles in HEPES solution, Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites were successfully synthesized by a two-step method, which showed excellent antibacterial properties against S. aureus. Furthermore, the Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites could be recycled and reused due to their superparamagnetic properties and retained their antibacterial activities.

---

1. Introduction

In the past decade, magnetite iron oxide nanostructures including Fe₃O₄ have attracted increasing attention in the biomedical field, due to their biocompatibility, higher stability in physiological conditions, and low cytotoxicity, which endows them with potential applications in targeted drug delivery,^1–3 magnetic resonance imaging (MRI),^4–7 multimodal and ultrasensitive detection of cancer cells.^8,9 Therefore, much effort has been applied to the development of synthetic strategies for hydrophilic and biocompatible Fe₃O₄ nanoparticles. To date, various synthetic methods, such as co-precipitation,¹⁰ sol–gel processing,¹¹ microemulsion technique,¹² solvothermal synthesis,^13,14 sonochemical approach¹⁵ and thermal decomposition of organic iron precursor,^16–19 have been applied to the synthesis of magnetite nanoparticles. Among them, variations in the molar ratio of Fe³⁺ and Fe²⁺ often lead to rather complicated changes in the crystalline structures of iron oxides,²⁰ while the products synthesized by a direct thermal decomposition route are generally soluble in organic solvents, which limits their applications in biomedical fields. As a matter of fact, it was a big challenge to maintain the coexistence of Fe²⁺ ions and Fe³⁺ ions with a fixed proportion (1 : 2) and prevent them from being reoxidized in the synthesis of Fe₃O₄ nanomaterials. Although purgation and protection with nitrogen^10,18 or argon^16,17 has been widely used. It still suffers from drawbacks such as high cost and complicated control processes. Therefore, it is of significance to explore a novel efficient, facile, and economic way to fabricate Fe₃O₄ nanostructures.

HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid) solution, as a convenient and environmental friendly buffer solution, is widely used in analytical, inorganic and biological studies.^21,22 Recently, HEPES solution was also used as an ideal reaction medium and precursor to prepare inorganic nanomaterials, including silver, gold, platinum metal nanoparticles and transition metal oxide (Co₃O₄, Mn₃O₄, and ZnO, etc.) nanomaterials with tunable morphology and size.^23–28

In our previous work, α-Fe₂O₃ nanostructures with controllable size and shape were successfully prepared in HEPES buffer solution,²⁹ in which HEPES acted as a stabilizer to prevent nanoparticles from aggregation. Inspired by the big challenge and previous work, we synthesized Fe₃O₄ nanomaterials from FeCl₂ precursor in HEPES buffer solution, without employing either purgation or protection procedure. The possible formation mechanism of Fe₃O₄ nanomaterials and the influence of inorganic anions on the morphologies of the Fe₃O₄ nanostructures were discussed. To the best of our knowledge, there are a few reports relevant to synthesis of Fe₃O₄ nanoparticles from a single Fe(II) precursor.^30,31 Besides, the fabrication of Fe₃O₄ nanostructures in HEPES buffer solution is also seldom reported. The magnetic properties and cytotoxicity of Fe₃O₄ nanoparticles towards HUVEC normal cells was also evaluated. Moreover, Au/Fe₃O₄ and Ag/Fe₃O₄ nanocomposites were also prepared in HEPES buffer solution and their enhanced antibacterial activities of Ag/Fe₃O₄ and Au/Fe₃O₄ against Staphylococcus aureus (S. aureus) were investigated. By utilizing superparamagnetic property of Fe₃O₄ nanospheres, the recyclability and stability of Au/Fe₃O₄ and Ag/Fe₃O₄ nanocomposites for the antibacterial test were also evaluated.

2. Experimental section

2.1 Chemicals

2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) was purchased from Sigma-Aldrich Chemical Company. Iron(II) chloride hydrate (FeCl₂·2H₂O), sodium nitrate (NaNO₃), sodium sulfate (Na₂SO₄), sodium bromide (NaBr), potassium iodide (NaI), sodium formate (HCOONa), and sodium acetate anhydrous (CH₃COONa) were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). All chemicals were directly used without further purification. The bacteria Staphylococcus aureus (S. aureus ATCC 9118) was provided by College of Life Science, Huazhong Normal University (Wuhan, China). The Luria-Bertani (LB) medium was used for for the growth of aerobic bacteria. The human umbilical vein endothelial cells (HUVEC) were supplied by College of Life Science, Wuhan University (Wuhan, China).

2.2 Synthesis

In a typical procedure, 15 mL FeCl₂ aqueous solution (200 mmol L⁻¹) was added into 15 mL HEPES aqueous solution (200 mmol L⁻¹, pH = 7.4) at room temperature. The mixed solution was stirred vigorously and transferred into a stainless steel autoclave with a Teflon liner. The autoclave was sealed and maintained at 150 °C for 12 h. After cooling down to room temperature, the black solid product was collected by centrifugation and washed with deionized water and alcohol for five times. The sample was finally dried in a vacuum desiccator at 80 °C for 12 h for further characterization. Other samples were also prepared under identical conditions by varying molar ratios of HEPES/FeCl₂, pH value of HEPES solution, reaction time, and inorganic additive. The detailed experimental parameters are listed in Table 1.

Table 1 Experimental Conditions for the Preparation of Fe₃O₄ Nanostructures

No.	Fe(II) precursor	Molar ratio^a	Time/h	T/°C	pH^b	Additive	Phase	Morphology
a HEPES/FeCl2 molar ratio.b pH value of HEPES solution.
S1	FeCl2	1 : 1	12	150	7.4	None	Fe3O4	Polyhedron
S2	FeCl2	0	12	150	7	None	α-Fe2O3	Polydispersed particles
S3	FeCl2	2 : 1	12	150	7.4	None	Fe3O4	Irregular polyhedron
S4	FeCl2	5 : 1	12	150	7.4	None	Fe3O4	Congeries
S5	FeCl2	1 : 1	12	150	5.2	None	α-Fe2O3	Polydispersed particles
S6	FeCl2	1 : 1	12	150	10.9	None	Fe3O4	Nanoparticles
S7	FeSO4	1 : 1	12	150	7.4	None	Fe3O4	Nanorods
S8	FeCl2	2 : 1	12	150	7.4	NaNO3	Fe3O4	Nanoparticles
S9	FeCl2	2 : 1	12	150	7.4	NaSO4	Fe3O4	Polyhedral
S10	FeCl2	2 : 1	12	150	7.4	NaBr	Fe3O4	Polyhedral
S11	FeCl2	2 : 1	12	150	7.4	NaI	Fe3O4	Nanoparticles
S12	FeCl2	2 : 1	12	150	7.4	HCOONa	Fe3O4	Polyhedra
S13	FeCl2	2 : 1	12	150	7.4	CH3COONa	Fe3O4	Spherical

---

For Au/Fe₃O₄ nanocomposites, 50 mg Fe₃O₄ nanoparticles (S15) was added to 10 mL HEPES buffer solution (50 mmol L⁻¹, pH 7.4) with vigorous stirring. Then, 1 mL HAuCl₄ solution (10 mmol L⁻¹) was introduced into the mixture and stirred vigorously for 2 h in the dark. The Au/Fe₃O₄ nanocomposites were collected by centrifugation and washed three times with distilled water and absolute ethanol. For Ag/Fe₃O₄ nanocomposites, 50 mg Fe₃O₄ nanoparticles (S15) was added to 10 mL HEPES buffer solution (50 mmol L⁻¹, pH 7.4) with vigorous stirring. Then, 1 mL AgNO₃ solution (10 mmol L⁻¹) was introduced into the mixture. After that, the reaction system was irradiated by UV light for 2 h under vigorous stirring. Finally, Ag/Fe₃O₄ nanocomposites were collected by centrifugation and washed three times with distilled water and absolute ethanol. The final Au/Fe₃O₄ and Ag/Fe₃O₄ products were stored in a desiccator for characterization and antibacterial activity studies.

2.3 Characterization

Power X-ray diffraction (XRD) was carried out on a Bruker AXS D8 Discover (Cu Kα = 1.5406 Å). The scanning rate is 1° min⁻¹ in the 2θ range from 10 to 80 degrees. The scanning electron microscopy (SEM) images were performed on a S4800 field emission scanning electron microscope (FESEM, Hitachi, Japan) at an accelerating voltage of 5 kV. The transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM) images and selected area electron diffraction (SAED) patterns were recorded on a Philips Tecnai G2 20 electron microscope at an accelerating voltage of 200 kV. TEM samples were prepared by dispersing some solid product into ethanol and then sonicating for approximately 30 seconds. A few drops of the suspension were deposited on copper grids, which were then put into the desiccators for drying. The energy-dispersive X-ray (EDX) analysis was performed on an Oxford Instruments INCA with a scanning range from 0 to 20 keV. The Raman spectra were measured at room temperature under the backscattering geometric configuration using WITec-Alpha confocal micro-Raman system. The excitation light was the 514.5 nm line of an Ar⁺ laser with 30 mW output power. The magnetic measurements were recorded on a SQUID magnetometer at 298 K, Quantum Design MPMS.

2.4 In vitro antibacterial activity and cytotoxicty assessment

The antibacterial activities of Ag/Fe₃O₄ and Au/Fe₃O₄ were tested using the antibacterial drop test against a gram positive bacterium (S. aureus). Suspension culture of the model organism S. aureus was cultured in Luria-Bertani (LB) medium at 37 °C for 24 h. The bacterial suspension was then washed and diluted to 5 × 10⁵ CFU mL⁻¹. 0.1 g Ag/Fe₃O₄ or Au/Fe₃O₄ nanocomposites was added into LB medium (10 mL) containing 5 × 10⁵ CFU mL⁻¹ S. aureus and then incubated at 37 °C on a rotary shaker for 18 h. Subsequently, aliquots (10 μL each) of diluted bacterial solution were spread over LB agar plates. After 24 h incubation at 37 °C, the number of bacterial colonies of surviving S. aureus was counted. The antibacterial activities of the recycled Ag/Fe₃O₄ or Au/Fe₃O₄ nanocomposites were also evaluated by same procedure. Additionally, 10 μL 5 × 10⁵ CFU mL⁻¹ of live S. aureus without treatment with Ag/Fe₃O₄ or Au/Fe₃O₄ nanocomposites was spread onto the LB agar plate and the number of colonies was counted as a control. The cytotoxicity study was carried out through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay toward HUVEC cells.

3. Results and discussion

3.1 Characterization of the polyhedron Fe₃O₄ nanoparticles

Fig. 1a shows the XRD pattern of the product prepared by hydrothermal method from FeCl₂ precursor in HEPES buffer solution with HEPES/FeCl₂ molar ratio of 1 : 1 (S1). Typically, all the diffraction peaks in Fig. 1a could be readily indexed to cubic phase Fe₃O₄, which is in good agreement with the standard data of Fe₃O₄ (JCPDS no. 01-1111). No other impurity peak was detected in this pattern, indicative of high purity of the product. Taking into account of both magnetite (Fe₃O₄) and maghemite (γ-Fe₂O₃) have a similar XRD pattern,³² Raman spectroscopy was further used to distinguish the crystal phase of the product. The Raman spectroscopy of sample S1 was shown in Fig. 1b, in which a characteristic intense absorption peak at 665 cm⁻¹ was observed, corresponding to typical A_1g model of Fe₃O₄,^33–35 confirming the formation of Fe₃O₄ by employing a HEPES-involved hydrothermal method.

Fig. 1 XRD pattern (a), Raman spectroscopy (b) and SEM images (c and d) synthesized by hydrothermal process at 150 °C for 12 h with 1 : 1 of HEPES/FeCl₂ mole ration.

SEM images (Fig. 1c and d) reveal that a large quantity of Fe₃O₄ nanoparticles was obtained in HEPES solution by hydrothermal method. It was observed that these nanoparticles were well-dispersed and had polyhedron morphology. The diameters of the Fe₃O₄ nanoparticles varied from 50 to 100 nm, and the average diameters estimated from SEM images observation was about 70 nm. EDX spectrum was also used to analyze the elemental compositions of the as-synthesized Fe₃O₄ nanoparticles. It illustrates that the sample is composed of Fe and O, which is in good agreement with the XRD result (Fig. S1, ESI†).

The morphology and structure of Fe₃O₄ nanoparticles (S1) was further characterized by TEM, HRTEM images, and SAED patterns. TEM images of Fig. 2a and b demonstrate that these nanoparticles are well-dispersed and have diameters of 50 to 100 nm. SAED pattern of sample S1 (Fig. 2c) displays several diffraction rings, which is the sum of diffraction patterns of different Fe₃O₄ nanoparticles. Each diffraction ring could be well indexed to the corresponding crystal planes of magnetite, indicative of good crystallinity of Fe₃O₄ nanoparticles. HRTEM image of a single Fe₃O₄ nanoparticle shown in Fig. 2d reveals clear lattice fringes, which also illustrates the highly crystalline nature of the as-synthesized Fe₃O₄ nanoparticles. A corresponding SAED pattern (inset of Fig. 2d) of a single nanoparticle was also examined, and the unique pattern of diffraction spots confirms a single crystalline structure of Fe₃O₄ nanoparticles.

Fig. 2 Typical TEM images (a and b), ED pattern (c) and HRTEM image (d) of sample S1. Inset of (d) is a SAED pattern of the selected single nanoparticle.

3.2 Function of HEPES

In the previous work, it was reported that HEPES could be used as a reducing agent in the fabrication of silver, gold and palladium nanoparticles,^23–25 as well as capping agent in the formation of metal oxide nanomaterials.²⁹ In the present reaction system, Fe²⁺ ions were not reduced to metallic iron in the HEPES solution, but partially were oxidized to Fe(III). However, when HEPES buffer solution was replaced by deionized water under identical conditions (S2), no Fe₃O₄ nanoparticles were prepared in the aqueous solution. Interestingly, the XRD pattern of the sample obtained in deionized water (S2) was in good agreement with the standard data of hexagonal α-Fe₂O₃ (JCPDS no. 84-0307) (Fig. 3a), indicating that the product was composed of α-Fe₂O₃. Fig. 3b shows the Raman spectroscopy of as-prepared product (S2), in which several typical modes of α-Fe₂O₃ including E_g(1), E_g(2), E_g(3), and A_1g(2) are observed, corresponding to five characteristics bands at 608, 496, 407, 243 and 223 cm⁻¹, respectively. It further demonstrates that the as-synthesized product (S2) belongs to pure α-Fe₂O₃ phase.

Fig. 3 XRD pattern (a), Raman spectroscopy (b) and SEM images (c and d) of the as-synthesized α-Fe₂O₃ particles (S2) in aqueous solution.

SEM images (Fig. 3c and d) reveal that α-Fe₂O₃ products have irregular nanoparticles morphology and a broad size distribution. The results indicate that HEPES played an important role in the fabrication of Fe₃O₄ nanoparticles. It was proposed that HEPES could act as a weak antioxidant to prevent the complete oxidation of Fe(II) to Fe(III) during the hydrothermal process. On the other hand, in this HEPES-involved system, –OH groups, SO₃²⁻ groups and –N– could absorb on certain crystal planes or chelate with Fe atoms as a competing agent to form steric hindrance, which could also prevent partial Fe²⁺ ions from being oxidization.³⁶ Besides, HEPES molecules adsorbed on the surface of Fe₃O₄ crystals could further influence the crystal growth. The adorable HEPES molecules on the surface of Fe₃O₄ as verified by FTIR spectrum of Fe₃O₄. Before FTIR measurement, the Fe₃O₄ nanoparticles (S1) were washed with absolute ethanol and deioned water for several times to remove remaining HEPES molecules. Compared with the FTIR spectrum of pure HEPES, the peaks at 3394, 2356, 2330, 1645 and 1062 cm⁻¹ in the IR spectrum of Fe₃O₄ nanoparticles (S1) could be assignable to corresponding stretching vibrations of HEPES molecules, indicating of the existence of HEPES molecules on the surface of Fe₃O₄ nanoparticles (Fig. S2, ESI†). The strong absorption bands at 572 cm⁻¹ are assigned to Fe–O stretching vibration modes, being one of the characteristic adsorption bands of Fe₃O₄.³⁷ The disappearance of peak at 1180 cm⁻¹ assigning to the SO₃²⁻ vibration of HEPES might be due to the formation of O–Fe band. It suggests that the surface of the as-synthesized Fe₃O₄ nanoparticles were covered with HEPES molecules, which could alter the resistance of each facet, resulting in an isotropic growth of Fe₃O₄ seeds.

However, the excessive HEPES molecules adsorbed on the surface of Fe₃O₄ nanoparticles has a significant influence on the disparity and particle size of Fe₃O₄ nanocrystals. When the HEPES/FeCl₂ molar ratio was increased to 2, irregular polyhedron Fe₃O₄ nanostructures were obtained (S3), as shown in Fig. S3a (ESI†). These irregular Fe₃O₄ nanoparticles were aggregated and the diameters varied from 40 to 500 nm, having a broad size distribution. When the HEPES/FeCl₂ molar ratio was further increased to 5 (S4), the congeries of Fe₃O₄ nanoparticles with the average diameter of ∼300 nm were obtained (Fig. S3b, ESI†). The results illustrate that the diameter of Fe₃O₄ nanoparticles increased with the increase of HEPES/FeCl₂ molar ratio, which might be due to the excessive HEPES molecules absorbed on the surface of Fe₃O₄ nanoparticles.

Noticeably, the antioxidant capability of HEPES could be tuned by changing pH value of the reaction system, which has an influence on the fabrication of Fe₃O₄ nanoparticles. When the pH value of HEPES solution was 5.2 (S5), only hematite α-Fe₂O₃ nanoparticles (JCPDS no. 84-0307) with a broad size distribution from 0.1 to 1 μm were obtained (Fig. S4a and b, ESI†). However, when the pH value of HEPES solution was adjusted to 10.9, uniform Fe₃O₄ nanoparticles (S6) with an average size of 40 nm were fabricated (Fig. S4c and d, ESI†), which is smaller than that of the product prepared in HEPES solution with pH value of 7.4. The results indicate that pH value of HEPES solution not only could influence the oxidation state of final products but also the control particle size of Fe₃O₄. It was proposed that the acidic solution could accelerate the oxidation of Fe²⁺ to Fe³⁺ ions at low pH value.³⁸ While at high pH value, free OH⁻ is beneficial for the formation of Fe₃O₄ products, because the alkalescent HEPES solution could not only control the concentration of OH⁻, but also alter the concentration of Fe²⁺ ions via playing the role of complex agents.

3.3 Possible formation process

On the basis of the experimental results, it was proposed that the formation process of Fe₃O₄ nanoparticles involved the following steps. In the HEPES-involved reaction system, the weak alkali environment could provide small amount of OH⁻ ions. Fe(HEPES)_m(OH)₂ nanoclusters were formed through the coordination of Fe²⁺ ions with OH⁻ and HEPES molecules. Then, part of Fe (HEPES)_m(OH)₂ would react with the oxygen in the air and solution, gradually being oxidized to generate Fe³⁺ ions. Once Fe²⁺ and Fe³⁺ were coexisted with OH⁻ in the reaction system, Fe₃O₄ nanocrystals will be quickly formed. At the same time, free HEPES molecules could act as an antioxidant and a capping agent in the formation of Fe₃O₄ nanoparticles. The morphologies and sizes of Fe₃O₄ nanocrystals could be tailored by changing the amounts of HEPES and the pH value of the reaction system. As a weak reductant, excessive HEPES molecules could prevent the fully oxidation of Fe²⁺ to Fe³⁺. On the other hand, HEPES presents different forms under different pH value, leading to different combination of HEPES molecule and Fe₃O₄ nuclei. The possible reaction processes in aqueous solution can be summarized in equations below (1)–(3):

Fe²⁺ + m HEPES + 2OH⁻ → Fe(HEPES)_m(OH)₂ (1)

4 Fe(HEPES)_m(OH)₂ + O₂ + 2H₂O → 4Fe³⁺ + 12OH⁻ + 4m HEPES (2)

Fe²⁺ + 2Fe³⁺ + 8OH⁻ → Fe₃O₄ + 4H₂O (3)

3.4 Influence of inorganic anions

Interestingly, it was found that Fe₃O₄ nanorods were fabricated when FeSO₄ was used as iron precursor, insteading of FeCl₂. Fig. 4a shows the low and high-magnification SEM images of Fe₃O₄ product prepared from FeSO₄ (S7), revealing that buddles of Fe₃O₄ nanorods were obtained. TEM image (Fig. 4b) shows that the nanorods are about 20–50 nm in diameter and 150–400 nm in length. SAED pattern of Fe₃O₄ nanorods (inset of Fig. 4b) displays several diffraction rings, which is the sum of the diffraction pattern of different individual nanorods, indicative of good crystallinity of the as-synthesized Fe₃O₄ nanorods. The results indicate that the morphologies of Fe₃O₄ nanostructures could be altered by using different iron precursors containing different inorganic anions, which plays an important role in the controlled synthesis of Fe₃O₄ nanocrystals.

Fig. 4 SEM (a) and TEM (b) images of the as-synthesized Fe₃O₄ nanorods by using FeSO₄ as the iron precursor (S7). Inset of Fig. 4(b) is the SAED pattern of sample S7.

To further systematically investigate the effects of inorganic anions on the morphologies of Fe₃O₄ nanocrystals, controlled experiments were carried out under the identical conditions by involving different inorganic salts, including NaNO₃, Na₂SO₄, NaBr, NaI, HCOONa and CH₃COONa (Table 1). As shown in Fig. 5a, uniform Fe₃O₄ nanoparticles with an average diameter of about 100 nm were obtained in the presence of NaNO₃ (S8). HRTEM image (Fig. 5b) of a single nanoparticle reveals the well-resolved interplanar d-spacing of 0.30 nm, which corresponds to the (220) lattice planes. The corresponding SAED pattern (inset of Fig. 5b) shows a unique pattern of diffraction spots, confirming its single crystal structure. Fig. 5c and d show SEM images of the products obtained with the addition of Na₂SO₄ (S9). It reveals that the product is mainly composed of polyhedral Fe₃O₄ nanocrystals with a diameter ranging 100 to 800 nm. When NaBr was involved in the synthesis, relatively uniform polyhedral Fe₃O₄ nanocrystals with an average diameter of 100 nm were obtained, which shows a narrow size distribution (S10, Fig. 5e and f). Meanwhile, SAED pattern of a single nanocrystal (insert of Fig. 5f) demonstrates that the Fe₃O₄ nanocrystal is a single crystal. In the presence of NaI, it was observed that aggregated Fe₃O₄ nanoparticles with a broad size distribution were obtained (S11, Fig. 5g and h). When HCOONa was introduced to the reaction system, uniform polyhedral Fe₃O₄ nanocrystals with an average diameter of 100 nm were formed (S12, Fig. 5i and j). SAED pattern of sample S12 reveals several diffraction rings, indicative of well-crystalline nature. Importantly, when CH₃COONa was instead of HCOONa in this synthesis, the product was mainly composed of uniform spherical Fe₃O₄ nanoparticles with an average diameter of 50 nm (S13, Fig. 5k and l). TEM image clearly reveals the spherical morphology of sample S13. The clear diffraction spots in SAED pattern of a single nanoparticle could readily be indexed to (111) and (220), which confirmed that the nanoparticle was well-crystallized. HRTEM image (inset of Fig. 5l) reveals a clear lattice fringe with d-spacing of 0.49 nm, corresponding to the interplanar d-spacing of (111).

Fig. 5 SEM and TEM images of the product obtained by HEPES buffer solution at 150 °C for 12 h when different inorganic salts (4 mmol) were introduced into this mixture (a and b) NaNO₃ (S8), (c and d) Na₂SO₄ (S9), (e and f) NaBr (S10), (g and h) NaI (S11), (i and j) HCOONa (S12), (k and l) CH₃COONa (S13).

Based on the experiment results, it was concluded that the morphologies of the Fe₃O₄ products were closely related to the anions involved in the solution. Generally speaking, it has a strong tendency to agglomerate during the formation of magnetic nanoparticles in the liquid phase. In this synthesis, it was proposed that the inorganic anions could act as a promoter for electrostatic stabilization, which can prevent the agglomeration of nanocrystals during the nucleation and growth. Similar results were also reported by Cao et al. and Deng et al. in the synthesis of Fe₃O₄ microspheres when NaCl and CH₃COONa were used.^39,40

3.5 Magnetic and in vitro cytotoxicity properties of Fe₃O₄ nanoparticles

The magnetic properties of the as-synthesized Fe₃O₄ nanoparticles (S1, S3, and S13) were evaluated by using a superconducting quantum interference device (SQUID) magnetometer at room temperature. Fig. 6a shows the magnetic hysteresis loop of different Fe₃O₄ nanoparticles, and the measured magnetic saturation values (M_s) are 75.0, 80.7, and 86.6 emu g⁻¹ for S1, S13 and S3, respectively. Compared with samples S1 and S3, high saturation magnetization was detected when the particle size increased. It was in good agreement with the previous reports that the magnetization of small particles decreases as the particle size decreases.⁴¹ On the other hand, it was also reported that magnetic oxidation could cause a decrease in saturation magnetization.⁴² In this work, a higher mole ratio of HEPES/FeCl₂ resulted in the coating of more HEPES molecules on the surface of Fe₃O₄ nanoparticles (S3), which could prohibit its oxidization, thus reading to the increase of M_s. In addition, compared with many organics-coated Fe₃O₄ nanocrystals synthesized by conventional aqueous solution routes,^43–45 it was found the Fe₃O₄ nanocrystals prepared in this work generally showed a better magnetization, which implies the potential of current approach toward synthesis advantages of highly crystallized, uniform, and magnetized iron oxide nanoparticles.

Fig. 6 (a) Hysteresis loop of the as-prepared Fe₃O₄ nanoparticles measured at 300 K (S1; S3; S13); (b) cytotoxicity of spherical Fe₃O₄ nanoparticles (S13) towards HUVEC cells at the concentrations of 5–100 μg mL⁻¹ after 24 h incubation.

The good magnetic property endows Fe₃O₄ nanoparticles with potential biological application. Hence, the cytotoxicity of the as-synthesized Fe₃O₄ nanoparticles (S13) towards HUVEC normal cells was investigated by MTT assay. Fig. 6b shows the viability of HUVEC cells after 24 h incubation with different concentrations of Fe₃O₄ nanoparticles (S13). It was obviously observed that the spherical Fe₃O₄ nanoparticles (S13) exhibited a negligible cytotoxic profile even at the concentration of 100 μg mL⁻¹. It illustrates that the Fe₃O₄ nanoparticles displayed none cytotoxicity towards the growth of HUVEC normal cells, indicative of good biocompatibility and promising biological applications.

3.6 Noble metal (Au, Ag)/Fe₃O₄ nanocomposites

To explore the biological application of Fe₃O₄ nanoparticles, Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites prepared via a two-step method. The morphology and structure of obtained Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites were characterized by TEM images. Fig. 7a and b show TEM images of the as-prepared Ag/Fe₃O₄ nanocomposites, which reveals that large quantities of silver nanoparticles were deposited on the surface of Fe₃O₄ nanocrystals. The diameters of Ag nanoparticles varied from 20 to 40 nm. EDX analysis of Ag/Fe₃O₄ nanocomposites confirms its elemental compositions of the prepared composites, which illustrates that the composite is composed of Fe, O, and Ag, indicative of the formation of Ag/Fe₃O₄ composites. Employing a similar route, Au/Fe₃O₄ nanocomposites were also successfully prepared. The amount of Ag and Au nanoparticles in the composites is 3.49% (Atomic%) and 1.69% (Atomic%), respectively. As shown in Fig. 7d and e, Au nanoparticles with diameters of 10–20 nm were deposited on the surface of Fe₃O₄ nanocrystals. EDX spectrum shown in Fig. 7f also confirms that the elemental composition of Au, Fe, and O in the Au/Fe₃O₄ nanocomposites.

Fig. 7 TEM images and EDX spectrum of Ag/Fe₃O₄ (a–c) and Au/Fe₃O₄ (d–f) nanocomposites obtained in HEPES buffer solution.

The magnetic properties of the as-prepared Au/Fe₃O₄ and Ag/Fe₃O₄ nanocomposites were also investigated. As depicted in Fig. 8a, the magnetic hysteresis measurements of Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites was carried out in applied magnetic field at 300 K. It was found that the coercively force of these two nanocomposites was almost negligible at 300 K, indicative of the superparamagnetic property of Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites at room temperature. The saturation magnetization (M_s) of Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites was about 73 and 67 emu g⁻¹, respectively, illustrating a little decrease compared with that of pure Fe₃O₄ nanocrystals (S13, 80 emu g⁻¹). The decrease in magnetization was not only due to mass decreasing of Fe₃O₄ nanocrystals resulted from the involving of silver and gold nanoparticle but also the diamagnetic contribution of the Au and Ag nanoparticles coating on the surface of Fe₃O₄ nanocrystals.⁴⁶ However, Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites still remained strong magnetization, which could have potential applications in bacterial disinfect, protein separation, photothermal therapeutics, catalysis, biological sensing, and so on. For example, it displayed ideal recycle properties in the antibacterial test by utilizing its strong magnetization, as illustrated in Fig. 8b.

Fig. 8 (a) Room temperature magnetization curves of Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites; (b) the schematic illustration of the antibacterial activities for recycled of Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites.

3.7 Antibacterial activities of Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites

It is well known that many metal nanoparticles exhibited intrinsic antibacterial properties, as well as pose potential toxicity to human cells.^47–49 Hence, to develop safe antibiotics, modification and isolation of metal nanoparticles is of great significance. In this work, the antibacterial activities against S. aureus of Fe₃O₄, Ag/Fe₃O₄ and Au/Fe₃O₄ were investigated by colony counting method. As shown in Fig. S5 (ESI†), no inhibition zone was observed in the disc cultured with S. aureus, indicating that bare Fe₃O₄ nanoparticles exhibited no antibacterial activity towards S. aureus. Fig. 9a–c show the bacterial growth in LB agar plates containing Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites, respectively. It was observed the CFU numbers decreased dramatically with addition of Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites into the S. aureus suspension, which indicates that Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites possess excellent antibacterial properties. Furthermore, Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites can be easily recycled due to their superparamagnetism property. The antibacterial properties of recycled Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites were evaluated by measuring their antibacterial rate. Fig. 9d shows the antibacterial rates of recycled Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites against S. aureus at different recycling times. It was observed that Ag/Fe₃O₄ nanocomposites remained excellent antibacterial property after five recycling times. The bacterial inhibition rates against S. aureus of Au/Fe₃O₄ nanocomposites shows a slightly decrease in the fourth cycle and obviously reduce to 40% after five recycling.

Fig. 9 (a–c) Agar plates of S. aureus bacterial growth in the absence and presence of Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites; (d) antibacterial rate of recycled Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites for different recycling times for S. aureus.

4. Conclusions

A facile and environment-friendly hydrothermal route has been employed to synthesize magnetite (Fe₃O₄) nanoparticles in HEPES solution. HEPES molecules played an important role in the fabrication of Fe₃O₄ nanoparticles. It was proposed that HEPES acted as a weak antioxidant to prevent from the complete oxidation of Fe(II) to Fe(III) in the formation of Fe₃O₄ nanocrystals. On the other hand, HEPES was also considered as a capping agent adsorbing on the surface of Fe₃O₄ nanoparticle to mediate the crystal growth and aggregation. The possible mechanism for the formation of Fe₃O₄ nanomaterials in HEPES solution was discussed. The influence of inorganic anions on the morphologies of Fe₃O₄ nanocrystals was also studied. The synthesized Fe₃O₄ nanoparticles exhibited nontoxicity towards HUVEC normal cells. Furthermore, Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites were successfully synthesized and showed excellent antibacterial ability against S. aureus. Meanwhile, the nanocomposite could be easily separated from solution by utilizing their superparamagnetic properties. Furthermore, Ag/Fe₃O₄ and Au/Fe₃O₄ nanocomposites still remained good antibacterial activities in the recycling usage. This work not only provides a novel strategy to prepare magnetite nanoparticles, but also indicates the potential biological applications of magnetic nanoparticles.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21371139), Key Program of Wuhan Science and Technology Bureau (201260523183) and High-Tech Industry Technology Innovation Team Training Program of Wuhan Science and Technology Bureau (2014070504020243).

References

1. J. Kim, J. E. Lee, S. H. Lee, J. H. Yu, J. H. Lee, T. G. Park and T. Hyeon, Adv. Mater., 2008, 20, 478–483.
2. J. H. Maeng, D.-H. Lee, K. H. Jung, Y.-H. Bae, I.-S. Park, S. Jeong, Y.-S. Jeon, C.-K. Shim, W. Kim, J. Kim, J. Lee, Y.-M. Lee, J.-H. Kim, W.-H. Kim and S.-S. Hong, Biomaterials, 2010, 31, 4995–5006.
3. J. E. Lee, N. Lee, H. Kim, J. Kim, S. H. Choi, J. H. Kim, T. Kim, I. C. Song, S. P. Park, W. K. Moon and T. Hyeon, J. Am. Chem. Soc., 2009, 132, 552–557.
4. J. Zhu, Y. Lu, Y. Li, J. Jiang, L. Cheng, Z. Liu, L. Guo, Y. Pan and H. Gu, Nanoscale, 2014, 6, 199–202.
5. X. Shi, S. H. Wang, S. D. Swanson, S. Ge, Z. Cao, M. E. Van Antwerp, K. J. Landmark and J. R. Baker, Adv. Mater., 2008, 20, 1671–1678.
6. L. Xiao, J. Li, D. F. Brougham, E. K. Fox, N. Feliu, A. Bushmelev, A. Schmidt, N. Mertens, F. Kiessling, M. Valldor, B. Fadeel and S. Mathur, ACS Nano, 2011, 5, 6315–6324.
7. N. Lee and T. Hyeon, Chem. Soc. Rev., 2012, 41, 2575–2589.
8. J. Liu, W. Zhang, H. Zhang, Z. Yang, T. Li, B. Wang, X. Huo, R. Wang and H. Chen, Chem. Commun., 2013, 49, 4938–4940.
9. L. Zhang, W.-F. Dong and H.-B. Sun, Nanoscale, 2013, 5, 7664–7684.
10. Y. S. Kang, S. Risbud, J. F. Rabolt and P. Stroeve, Chem. Mater., 1996, 8, 2209–2211.
11. Y. Lu, Y. Yin, B. T. Mayers and Y. Xia, Nano Lett., 2002, 2, 183–186.
12. Z. H. Zhou, J. Wang, X. Liu and H. S. O. Chan, J. Mater. Chem., 2001, 11, 1704–1709.
13. J. Lu, X. Jiao, D. Chen and W. Li, J. Phys. Chem. C, 2009, 113, 4012–4017.
14. A. Yan, X. Liu, G. Qiu, N. Zhang, R. Shi, R. Yi, M. Tang and R. Che, Solid State Commun., 2007, 144, 315–318.
15. R. V. Kumar, Y. Koltypin, X. N. Xu, Y. Yeshurun, A. Gedanken and I. Felner, J. Appl. Phys., 2001, 89, 6324–6328.
16. T. Hyeon, S. S. Lee, J. Park, Y. Chung and H. B. Na, J. Am. Chem. Soc., 2001, 123, 12798–12801.
17. J. Rockenberger, E. C. Scher and A. P. Alivisatos, J. Am. Chem. Soc., 1999, 121, 11595–11596.
18. S. Sun, H. Zeng, D. B. Robinson, S. Raoux, P. M. Rice, S. X. Wang and G. Li, J. Am. Chem. Soc., 2003, 126, 273–279.
19. F.-h. Lin, H.-H. Peng, Y.-H. Yang and R.-a. Doong, J. Nanopart. Res., 2013, 15, 1–13.
20. E. Tronc, P. Belleville, J. P. Jolivet and J. Livage, Langmuir, 1992, 8, 313–319.
21. N. E. Good, G. D. Winget, W. Winter, T. N. Connolly, S. Izawa and R. M. M. Singh, Biochemistry, 1966, 5, 467–477.
22. Q. Yu, A. Kandegedara, Y. Xu and D. B. Rorabacher, Anal. Biochem., 1997, 253, 50–56.
23. M.-H. So, C.-M. Ho, R. Chen and C.-M. Che, Chem.–Asian J., 2010, 5, 1322–1331.
24. R. Chen, J. Wu, H. Li, G. Cheng, Z. Lu and C.-M. Che, Rare Met., 2010, 29, 180–186.
25. R. W.-Y. Sun, R. Chen, N. P.-Y. Chung, C.-M. Ho, C.-L. S. Lin and C.-M. Che, Chem. Commun., 2005, 5059–5061.
26. J. Xie, J. Y. Lee and D. I. C. Wang, Chem. Mater., 2007, 19, 2823–2830.
27. H. Li, Z. Lu, J. Wu, H. Yu, X. Yu and R. Chen, Mater. Lett., 2010, 64, 1939–1942.
28. Q. Li, E.-t. Liu, Z. Lu, H. Yang and R. Chen, Mater. Lett., 2014, 130, 115–119.
29. H. Li, Z. Lu, Q. Li, M.-H. So, C.-M. Che and R. Chen, Chem.–Asian J., 2011, 6, 2320–2331.
30. X. Zhang, X. Xu, T. Li, M. Lin, X. Lin, H. Zhang, H. Sun and B. Yang, ACS Appl. Mater. Interfaces, 2014, 6, 14552–14561.
31. T. Wang, L. Zhang, H. Wang, W. Yang, Y. Fu, W. Zhou, W. Yu, K. Xiang, Z. Su, S. Dai and L. Chai, ACS Appl. Mater. Interfaces, 2013, 5, 12449–12459.
32. N. Pinna, S. Grancharov, P. Beato, P. Bonville, M. Antonietti and M. Niederberger, Chem. Mater., 2005, 17, 3044–3049.
33. D. Bersani, P. P. Lottici and A. Montenero, J. Raman Spectrosc., 1999, 30, 355–360.
34. D. L. A. de Faria, S. Venâncio Silva and M. T. de Oliveira, J. Raman Spectrosc., 1997, 28, 873–878.
35. J. Zhang, H. Ji, Y. Wei, Y. Wang and N. Wu, J. Phys. Chem. C, 2008, 112, 10688–10691.
36. R. Nakon and C. Krishnamoorthy, Science, 1983, 221, 749–750.
37. H. Tang, B. J. Kwon, J. Kim and J.-Y. Park, J. Phys. Chem. C, 2010, 114, 21366–21370.
38. X. Yang and N. D. Chasteen, Biochem. J., 1999, 338, 615–618.
39. S.-W. Cao and Y.-J. Zhu, Nanoscale Res. Lett., 2011, 6, 1–7.
40. H. Deng, X. Li, Q. Peng, X. Wang, J. Chen and Y. Li, Angew. Chem., Int. Ed., 2005, 44, 2782–2785.
41. S.-W. Cao, Y.-J. Zhu and J. Chang, New J. Chem., 2008, 32, 1526–1530.
42. R. L. Rebodos and P. J. Vikesland, Langmuir, 2010, 26, 16745–16753.
43. K. Tao, H. Dou and K. Sun, Chem. Mater., 2006, 18, 5273–5278.
44. X. Liang, X. Wang, J. Zhuang, Y. Chen, D. Wang and Y. Li, Adv. Funct. Mater., 2006, 16, 1805–1813.
45. J. Sun, S. Zhou, P. Hou, Y. Yang, J. Weng, X. Li and M. Li, J. Biomed. Mater. Res., Part A, 2007, 80A, 333–341.
46. Y. Zhai, J. Zhai, Y. Wang, S. Guo, W. Ren and S. Dong, J. Phys. Chem. C, 2009, 113, 7009–7014.
47. I. Sondi and B. Salopek-Sondi, J. Colloid Interface Sci., 2004, 275, 177–182.
48. M. Rai, A. Yadav and A. Gade, Biotechnol. Adv., 2009, 27, 76–83.
49. N. Khlebtsov and L. Dykman, Chem. Soc. Rev., 2011, 40, 1647–1671.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra12536c

---