Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226 www.elsevier.com/locate/colsurfa Preparation and characterization of magnetite nanoparticles coated by amino silane Ming Ma
, Yu Zhang, Wei Yu, Hao-ying Shen, Hai-qian Zhang, Ning Gu National Laboratory of Molecular and Biomolecular Electronics, Southeast University, Nanjing 210096, PR China Received 13 January 2002; received in revised form 21 May 2002; accepted 18 June 2002 Abstract Magnetite nanoparticles were prepared by coprecipitation of Fe2 and Fe3 with NH4OH, and then, amino silane was coated onto the surface of the magnetite nanoparticles. Transmission electronic microscopy shows the average size of 7.5 nm in diameter. Powder X-ray diffraction and electronic diffraction measurements show the spinel structure for the magnetite nanoparticles. FT /IR spectra indicate that amino silane molecules have been bound onto the surface of the magnetite nanoparticles by Fe/O/Si chemical bonds. Energy dispersive X-ray spectroscopy (SEM /EDS) indicates atomic ratio of 96.75:3.25 for Fe:Si, implying a nearly monolayer coating of amino silane on the magnetite particle surface according to a rough calculation. By an enzyme-linked assay, it was proved that the amino silane-coated magnetite nanoparticles could significantly improve the protein immobilization. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Magnetite nanoparticles; Core-shell structure; Surface coating; Amino silane; Protein immobilization 1. Introduction Magnetic particles (microspheres, nanospheres and ferrofluids) are widely studied for their applications in various fields in biology and medicine such as enzyme and protein immobilization, genes, radiopharmaceuticals, magnetic resonance imaging MRI, diagnostics, immunoassays, RNA and DNA purification, magnetic cell separation and purification, magnetically controlled transport of anti-cancer drugs as well as hyperthermia generation [1 /3]. These magnetic beads are
Corresponding author E-mail address: maming@seu.edu.cn (M. Ma). generally of core /shell type: biological species cells, nucleic acids, proteins are connected to the magnetic core through an organic or polymeric shell. The shells are either biocompatible in general (such as dextran, PEG, etc.), or possessing active groups which can be conjugated to biomolecules such as proteins and enzymes. Therefore, the investigation of magnetic nanoparticles with organic coating is of significance for applications. In this work we prepared magnetite nanoparticles coated with a near monolayer of amino silane, which has active group of /NH2 that can connect biomolecules, drugs and so on. And the morphology, structure and composition of the coated magnetite nanoparticles were characterized by TEM, ED, XRD, FT-IR and SEM /EDS. 0927-7757/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 2 ) 0 0 3 0 5 - 9 220 M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226 Furthermore, to prove that amina silane-coated magnetite nanoparticles can be conjugated to some biomolecule, an enzyme-linked colorimetric assay was carried out after the enzyme horseradish peroxidase (HRP) of different concentrations was used to interact with the coated and the uncoated magnetite nanoparticles. magnetite particles were measured by energy dispersive X-ray spectroscopy (SEM /EDS, EDAX, PV9100). Fourier transform infrared spectroscopy (FT-IR, Nicolet, 750) of the samples were used to study the chemical bonds between Fe3O4 and APTS. 2.4. HRP-immobilized and its activity assays 2. Experimental 2.1. Synthesis of magnetite nanoparticles Magnetite was made according to the method of Molday [4]. Typically, a solution of mixture of FeCl3 (0.01 M) and FeSO4 (0.006 M) at pH 1.7 was prepared under N2 protecting. Then, ammonia aqueous solution (1.5 M) was dropped into it with violently stirring until the pH of the solution raised to 9. The obtained magnetite was washed immediately with water for 5 times and ethanol for 2 times by magnetic separation. Finally, part of magnetite nanoparticles were dispersed in ethanol with concentration of 0.0128 M, and the others were dried into powder at room temperature under vacuum. 2.2. APTS-coated magnetite nanoparticles 25 ml magnetite colloid ethanol solution prepared above was diluted to 150 ml by ethanol and 1 ml H2O. The solution was then treated by ultrasonic wave for 30 min. 35 ml 3-aminopropyltriethoxysilane ( NH2(CH2)3Si(OC2H5)3, APTS) was added into it with rapid stirring for 7 h. The result solution was washed with ethanol for 5 times, and then dried into powder at room temperature under vacuum. 2.3. Characterization The particle size and morphology of the samples were determined by transmission electronic microscopy (TEM, JEOL, JEM-200CX, 200 kV). Powder X-ray diffraction (XRD, Rigaku, D/Max-RA, Cu Ka) and electronic diffraction ( ED) were used to determine the crystal structure of the samples. The elemental analysis and APTS loading on The APTS-coated magnetite nanoparticles and uncoated magnetite nanoparticles as control were all dispersed in phosphate-buffered saline (PBS, 0.01 M, pH 7.4) with identical concentrations of 2 g l 1. Enzyme horseradish peroxidase HRP of different concentrations were added into 200 ml magnetite-PBS solutions. The mixtures were incubated in 37 8C for 1 h, and then retracted in 4 8C refrigeratory for 4 h. Then the mixtures were washed carefully by PBS for 4 times and shifted to other vessels to remove the dissociated enzymes. Developed by addition of substrates, namely 3,3?,5,5?-tetramethylbenzidine and hydrogen peroxide (TMB-H2O2), for 10 min, the reaction was stopped by 2 mol l 1 H2SO4. The optical density at 450 nm was read immediately in an automatic plate reader (Stat fax-2100, Beiken Company). All samples were tested in duplicate, arithmetic means and standard deviations of absorbance values were calculated (x9/s). 3. Results and discussion Fig. 1 is the TEM and ED photography of the magnetite nanoparticles coated with APTS, which shows that most of the particles are quasi-spherical and with an average diameter of 7.5 nm. The distribution of particle diameters is shown in Fig. 2. According to the ED pattern, the d -spacing can be calculated in the following eqution [5], LldR (1) where L is the distance between the test sample and the film ( L /137 cm), l is the wavelength of electron beam (l /0.0251 Å), R is the radius of the diffraction ring. The calculation results are shown in Table 1, which accord with the XRD M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226 Fig. 1. TEM and ED photography of the magnetite nanoparticles coated with APTS. pattern of the sample shown in Fig. 3 and indicate the inverse cubic spinel structure of Fe3O4. Compared with the theoretical values, the reduction of d -spacing obtained experimentally is due to lattice constrictions for nanosized particles [7]. Since there are large surface-to-volume atomic ratio, high surface activity, and amount of dangling bonds on nanoparticle surface, the atoms on the surface are apt to adsorb ions or molecules in solution. For Fe3O4 nanoparticles dispersed in a neutral aqueous solution, the bare atoms of Fe and O on the particle surface would adsorb OH  and H  respectively, so that there is OH-rich surface. The /OH on the surface can react with APTS as the process shown in Fig. 4. Therefore, the magnetite nanoparticles can be coated with APTS molecules by chemical bond. The fact was proven by comparison of FT-IR spectra of the coated and uncoated Fe3O4 nanoparticles shown in Fig. 5. It can be seen that, compared with the uncoated sample, the coated Fe3O4 nanoparticles 221 Fig. 2. Diameter distribution of magnetite nanoparticles coated with APTS. possess absorption bands in 2971.8 and 2925.5 cm 1 due to stretching vibration of C /H bond, band in 1091.5 cm 1 due to the stretching vibration of C /N bond, band in 1051.0 cm 1 due to the stretching vibration of Si /O bond, band in 885.2 cm 1 due to the bending vibration of /NH2 group. All of these reveal the existence of APTS. In addition, in Fig. 5(a) and (b) the absorption bands near 3400 and 1630 cm 1 refer to the vibration of remainder H2O in the samples. And there also exists the contribution of /NH2 for the band near 3400 cm 1 in Fig. 5(a). Previously, it was reported that the characteristic absorption bands of the Fe /O bond of bulk Fe3O4 were in 570 and 375 cm 1 [8]. However, in Fig. 5(b) these two bands shift to high wavenumbers of about 600 and 440 cm 1 respectively, and the band near 600 cm 1 is split into two peaks of 631.4 and 582.9 cm 1. A principal effect of finite size of nanoparticles is the breaking of a large number of bonds for surface atoms, resulting in the rearrangement of inlocalized electrons on the 222 M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226 Table 1 ED and XRD data for the magnetite nanoparticles coated with APTS R (cm) ED results-d (Å) XRD results-d (Å) Theory values-d (Å) Crystalline plane (hlk) [6] 1 0.73 4.74 4.78 4.85 (111) 2 1.19 2.89 2.83 2.97 (220) 3 1.39 2.47 2.50 2.53 (311) 4 1.67 2.06 2.08 2.10 (400) 5 2.04 1.69 1.70 1.71 (422) Fig. 3. XRD pattern of the magnetite nanoparticles coated with APTS. Fig. 4. The procedure of the coating reaction of APTS with magnetite nanoparticles. 6 2.19 1.57 1.60 1.62 (511) 7 2.37 1.47 1.48 1.48 (440) M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226 Fig. 5. FT-IR spectra of the coated (a) and uncoated (b) magnetite nanoparticles. 223 224 M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226 Fig. 6. SEM /EDS elemental analysis of APTS-coated Fe3O4 nanoparticles on Au substrate. particle surface [9]. And the lattice constrictions have been observed as indicated in Table 1. As a result, the surface bond force constant increases as Fe3O4 is reduced to nanoscale dimension, so that the absorption bands of IR spectra shift to higher wavenumbers. So the blue-shift of absorption bands of the Fe /O bond of the Fe3O4 nanopar- ticles can be observed. In addition, the split of the bands is attributed to the split of the energy levels of the quantized Fe3O4 nanoparticles[10]. It is also found that the characteristic absorption bands of the Fe /O bond of APTS-coated Fe3O4 shift to high wavenumbers of 636.4 and 590.1 cm 1 compared with that of uncoated Fig. 7. HRP-linked colorimetric assays of APTS-coated and uncoated Fe3O4 nanoparticles. M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226 Fe3O4(in 631.4 and 582.9 cm 1). The phenomenon can be explained according to the formation of Fe /O /Si bonds where Fe /O /H groups on the surface of the Fe3O4 particles are replaced by Fe / O /Si(O /)2 /R as shown in Fig. 4. More electronegativity of /Si(O /)2 / than H leads to the enhancement of bond force constant for Fe /O bonds[11], so that the absorption bands shift to high wavenumbers. Since we know the mean diameter of the magnetite nanoparticles is 7.5 nm, the number of Fe atoms in every Fe3O4 particle can be calculated by means of following formula, 4 NFe  3 pR3 Na V Fe3 O4 3 8945 (2) where V̄ Fe3 O4/refers to the molar volume of bulk Fe3O4, R is the mean radius of Fe3O4 nanoparticles, Na is Avogadro’s number. If there is a monolayer of APTS molecules coated on the Fe3O4 particle, the number of APTS molecules on the surface of every Fe3O4 nanoparticle can be calculated by following formula, NAPTS  SFe3 O4 SAPTS  4pR2 SAPTS 442 (3) where SFe3 O4 is the surface area of Fe3O4 particle, SAPTS is the area of surface coverage of about 40 Å2 per APTS molecule reported in the literature [12]. So the atomic ratio of Fe/Si is/NFe =NAPTS  20:2: Fig. 6 shows a typical SEM /EDS elemental analysis of APTS-coated magnetite nanoparticles. From the peak area of Fe and Si, the atomic ratio of Fe/Si is obtained to be 96.75/3.25 /30. This indicates that the surface APTS coverage ratio of Fe3O4 nanoparticles is about 67.3%. Probably, the incompleteness (a near monolayer) of the surface coating is owing to the incompleteness of surface hydroxylation and the existence of the spatial resistance for the surface coating reaction. Fig. 7 shows the result of the HRP-linked magnetite nanoparticles colorimetric assays. Obviously, the absorbances of HRP-linked APTScoated magnetite nanoparticles are higher than HRP-linked uncoated magnetite nanoparticles. It is revealed that the amount of adsorbed HRP on 225 the APTS-coated magnetite nanoparticles is 1.4 / 2.0 times higher than that of the uncoated magnetite nanoparticles according to the measured absorbance value. The increase of absorption is attributed to the contribution of APTS whose active group of /NH2 can be conjugated to HRP by chemical band, and the uncoated magnetite nanoparticles connect the HRP by static adsorption only. 4. Conclusions APTS-coated magnetite nanoparticles with 7.5 nm average diameter were prepared and characterized by TEM, ED, XRD, FT-IR, and SEM / EDS. Especially, FT-IR spectra were utilized to prove the formation of Fe /O /Si chemical bonds. A near monolayer APTS-coating on the particle surface was also indicated according to the comparison of the experimental analysis by SEM / EDS with a simple calculation. By an enzyme-linked assay, it has been proved that these APTS-coated magnetite nanoparticles could significantly improve the protein immobilization. Acknowledgements This work was supported by the National Natural Science Foundation of China (No.69890220, No.60171005) and the High Technology Research Subject of Jiangsu Province in China (BG2001006). I am also very grateful to Prof. Hong Jian-min of Center of Analysis and Test, Nanjing University for his helping in TEM experiments. References [1] U. Häfeli, W. Schütt, J. Teller, M. 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