A synthesis of polyethylene glycol (PEG)-coated magnetite Fe3O4 nanoparticles and their characteristics for enhancement of biosensor

The magnetite Fe3O4 nanoparticles were synthesized by using chemical co-precipitation method and these nanoparticles were successfully coated by polyethylene glycol (PEG) with variation concentrations of PEG. The magnetite Fe3O4 nanoparticles used as a bimolecular label (nano-tags), exhibiting a soft magnetic behavior with magnetization (Ms) of 77.16 emu g−1 and coercivity (Hc) of 50 Oe respectively. The polyethylene glycol (PEG) was used as a biocompatible polymer. The x-ray diffraction (XRD) patterns of the Fe3O4 showed that Fe3O4 was well crystallized. It also confirmed the existence of invers spinel. The diffraction peak of 35.4° was used to calculate the crystallite size. The estimation of Fe3O4 average crystallite size is 12 nm, while the PEG-coated Fe3O4 nanoparticles is 8.6 nm. The transmission electron microscopy (TEM) images of Fe3O4 showed that the morphology of magnetite Fe3O4 nanoparticle is spherical in shape with uniform grain size and good dispersibility despite the agglomeration it found in some place. The addition of PEG can decrease the agglomeration and reduce the particle size. The existence of PEG layer on Fe3O4 was confirmed by Fourier transform infrared (FTIR) spectroscopy. The result of Vibrating Sample Magnetometer (VSM) showed that saturation magnetization (Ms) of Fe3O4 nanoparticles decreased from 77.16 to 37.15 emu g−1 with the increase of PEG weight from 0% to 50%. Such Fe3O4 nanoparticles with favorable size and tunable magnetic properties are promising biosensor applications.


Introduction
In recent years, magnetic nanoparticles especially magnetite Fe 3 O 4 nanoparticles have been widely used in biomedical applications for collection and separation of bioactive molecules, targeted drug delivery, and biolabeling because of its characteristics, such as superparamagnetic, responses to biomolecules, biomoleculedispersions and high magnetic saturation (M s ) [1][2][3]. To be applied as a biosensor, magnetite Fe 3 O 4 nanoparticles must have good dispersibility in aqueous media and narrow size distribution. This is to avoid agglomeration in Fe 3 O 4 nanoparticles. However, pure magnetic nanoparticles tend to agglomerate as result of strong magnetic interactions. Therefore, for biomedical applications, one important problem to consider in the use of Fe 3 O 4 nanoparticles is stabilization. To increase their stabilization, Fe 3 O 4 nanoparticles have been stabilized by the formation of polymer layers on the surface of magnetic nanoparticles, using dextran, gen, cell, poly (vinyl alcohol) (PVA), enzyme, protein, gelatin, etc [4][5][6][7][8][9].
The use of magnetic nanoparticles to biological applications has two major problems. The first one, as what has been stated previously is easy agglomeration, chemical reactivity and high surface energy, especially when they are used as biological agents without being matched with proper characterization. For this reason, surface modification or coating method of magnetic nanoparticles is required with biocompatible polymer molecules. Polyethylene glycol (PEG) is the most popular and widely used biocompatible polymers because of its benefits such as increasing dispersibility, biocompatible, non-toxic, and low cost.
The co-precipitation method of iron salts is the simplest and most efficient chemical pathway to synthesis magnetic nanoparticle [10]. In this study, PEG-coated Fe 3 O 4 was synthesized following a simple two steps co-precipitation approach with various PEG concentrations. The synthesized PEG-coated Fe 3  were investigated by x-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and vibrating sample magnetometer (VSM).

Experiment details
The synthesis was carried out by chemical co-precipitation method by following the procedure. Synthesis of Fe 3 O 4 nanoparticles was obtained from the basic ingredients of dissolved hydrate compounds, namely ferric chloride hexahydrate (FeCl 3 .6H 2 O) and heptahydrate (FeSO 4 .7H 2 O) (From Merck, Germany) which are providers of Fe 3+ and Fe 2+ ions with a mass ratio of 8.109 g and 4.170 g respectively as shown in figure 1. All the materials had analytical purity and were used without further purification. The dissolution process uses 30 ml of distilled water. It was stirred for 15 min until the mixture is homogeneous. Next, 60 ml of 10% ammonia (NH 4 OH) solution was added dropwise and stirred using a magnetic stirrer at a speed of 450 rpm and a temperature of 60°C for 90 min. The result of the Fe 3 O 4 solution was washed using distilled water until the NH 4 OH odor disappeared. Next, the Fe 3 O 4 decantation process was carried out to obtain samples in the form of sediment. This deposition used the effect of the magnetic field from a permanent magnet to be more efficient. The precipitate is washed 7 times to minimize the amount of salt from other reactions dissolved in the sample [11]. The Fe 3 O 4 sample was dried in a furnace for 2 h at a temperature of 80°C. The relevant chemical reaction can be expressed as follows: The phase identification of Fe 3 O 4 and PEG-coated Fe 3 O 4 and crystallite size determination were done by x-ray diffractometer Shimazu XD equipped with a crystal monochromator employing Cu-Kα radiation of wavelength 1.5406 Å [12]. The coating and surfactant were only done for observing the functional groups by using Fourier transform infrared (FTIR) spectroscopy [13]. Magnetic characterization was carried out by Riken Denshi Co. Ltd. Vibrating sample magnetometer (VSM) under external magnetic fields up to ±15000 Oe at  room temperature [14]. The size and morphology were investigated by means of transmission electron microscopy (TEM) using JEOL JEM-1400 [15].

Result and discussion
Phase investigation of the crystalline products were conducted using XRD and patterns are presented in figure 2.
It is found that the d-spacing values of significant peaks match well with the data from JCPDS card (19-029) for Fe 3 O 4 nanoparticles. The diffraction peaks at 2θ can be assigned to the (220) (311) (400) (511) (440) confirmed the existence of invers spinel [16]. The determination of average crystallite size of Fe 3 O 4 is based on XRD line broadening and calculated using Scherrer's equation as follows: Based on the equation above, d is the average of crystallite size, 0.9 is the Scherrer constant, λ is the x-ray diffraction wavelength (λ=1.5406 Å), β is the full width at half maximum (FWHM) of plane (311) and θ is the Bragg angle in degree. The estimated diffraction peak is at the angle of 35.4°and the average crystallite size of Fe 3 O 4 is 12 nm and PEG-coated Fe 3 O 4 nanoparticles is 8.6 nm. In addition, another phase appeared on Fe 3 O 4 namely α-Fe 2 O 3 at 32°. This is due to the oxidation process that occurred in the synthesis process [17].  product. However, this peak was not observed in the Fe 3 O 4 spectrum. These results clearly show the surface modification of Fe 3 O 4 nanoparticles with PEG [18]. Figure 4(a) shows the magnetization curve of magnetite Fe 3 O 4 nanoparticles at room temperature. The maximum saturation magnetization (M s ) gain is 77.16 emu g −1 [19]. The magnetite Fe 3 O 4 nanoparticles exhibited good magnetic response. Other than that, it could be easily attracted by the near magnet with superparamagnetic behavior. The magnetization is related to particle size and cation distribution. The remanent magnetization (M r ) and coercivity field (H c ) gains are 7.65 emu g −1 and 50 Oe respectively. TEM images recorded from Fe 3 O 4 nanoparticles and PEG-coated Fe 3 O 4 nanoparticles are shown in figures 4(b) and (c) respectively. The morphology of magnetite Fe 3 O 4 nanoparticles is spherical with uniform grain size and good dispersibility despite the agglomeration found in some places. The addition of PEG could decrease the agglomeration and reduce the particle size. This is because PEG can modify the surface of Fe 3 O 4 particles so that the particle size is more monodisperse and uniform. In addition, lattice fringe patterns for both, are in good agreement with the XRD analysis, which corresponds to the crystal field (220), (311), (400), (440), and (511).
Other information obtained is the grain size distribution of Fe 3 O 4 nanoparticles and PEG-coated Fe 3 O 4 nanoparticles as shown in figure 5. Figure 5 shows the grain diameter distribution from TEM observations. The highest frequency shows the average size of grain diameter. The average grain diameter of Fe 3 O 4 nanoparticles shown in figure 5(a) is 14.5±0.5 nm whereas from figure 5(b) the diameter of PEG-coated Fe 3 O 4 nanoparticles is 12.5±0.5 nm. The good correlation between particle sizes obtained from Scherrer equation and TEM supported the highly crystalline structure as shown by XRD. From the results of the analysis, it can be concluded that PEG is able to control particle size and limit the crystal growth of Fe 3 O 4 .  Under room temperature, M-H hysteresis of PEG-coated Fe 3 O 4 nanoparticles was measured up to 15 kOe using a vibrating sample magnetometer (VSM). As shown in figure 6, all magnetization curves are shaped S-like over an external magnetic field, which is a typical superparamagnetic material, with zero coercivity in the M-H plot. Zero coercivity occurred because there was no residual magnetism when the external magnetic field was removed [20,21]. The saturation magnetization (M s ) of Fe 3 O 4 nanoparticles decreased from 77.16 to 37.15 emu g −1 with the increased PEG weight from 0 to 50% [22,23]. This is also supported by XRD analysis. This phenomenon shows that the greater the size of magnetite Fe 3 O 4 nanoparticles, the stronger the saturation magnetization [24]. Thus, the high magnetization indicated that PEG-coated Fe 3 O 4 nanoparticles have great potential for biomedical applications [25].

Conclusion
In this investigation, Fe 3 O 4 nanoparticles have been successfully synthesized by a PEG assisted co-precipitation method in which aqueous NH 4 OH solution was used as solvent agents. Formations of PEG-coated Fe 3 O 4 nanoparticles are confirmed by XRD and FTIR. TEM analysis confirmed that the physical size and average  crystallite size of the nanoparticles decreased with the increase of PEG weight. The prepared PEG-coated Fe 3 O 4 nanoparticles exhibited high saturation magnetization and superparamagnetic behavior which evidence multidomain behavior of the observed particles and the absence of coupling between the nanocrystals due to the presence of the PEG as polymer in the nanoparticles. Since many intrinsic properties of magnetic nanoparticles are size dependent, it can be concluded that the nanoparticles with different sizes will have important application in biomedical applications such as cancer diagnosis, targeted drug delivery, as well as biomolecular separations [26,27]. Therefore, the high saturation and superparamagnetic behavior of PEG-coated Fe 3 O 4 nanoparticle make it promising as biosensor applications.