In vitro cytotoxicity screening of water-dispersible metal oxide nanoparticles in human cell lines

In this study, we present in vitro cytotoxicity of iron oxide (Fe₃O₄) and manganese oxide (MnO) using live/dead cell assay, lactate dehydrogenase assay, and reactive oxygen species detection with variation of the concentration of nanoparticles (5–500 μg/ml), incubation time (18–96 h), and different human cell lines (lung adenocarcinoma, breast cancer cells, and glioblastoma cells). The surface of nanoparticles is modified with polyethyleneglycol-derivatized phospholipid to enhance the biocompatibility, water-solubility, and stability under an aqueous media. While the cytotoxic effect was negligible for 18 h incubation even at highest concentration of 500 μg/ml, MnO nanoparticle represented higher level of toxicity than those of Fe₃O₄ and the commercial medical contrast reagent, Feridex after 2 and 4 day incubation time. However, the cytotoxicity of Fe₃O₄ is equivalent or better than Feridex based on the live/dead cell viability assay. The engineered MnO and Fe₃O₄ exhibited excellent stability compared with Feridex for a prolonged incubation time.     

Chemically synthesized zinc finger molecules as nano-addressable probes for double-stranded DNAs

Magnetic nanoparticles (Fe₃O₄) were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The sizes and structure of the particles were characterized using transmission electron microscopy (TEM). The size of the particles was in the range between 9.7 and 56.4 nm. Cholesterol oxidase (CHO) was successfully bound to the particles via carbodiimide activation. FTIR spectroscopy was used to confirm the binding of CHO to the particles. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of the enzyme were significantly improved upon binding to the nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy for free and bound CHO was 13.6 and 9.3 kJ/mol, respectively. This indicated that the energy barrier of CHO activity was reduced upon binding onto Fe₃O₄ nanoparticles. The improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound enzyme. The study indicates that the stability and activity of CHO could be enhanced via attachment to magnetic nanoparticles and subsequently will contribute to better uses of this enzyme in various biological and clinical applications.     

Development of oleic acid-functionalized magnetite nanoparticles as hydrophobic probes for concentrating peptides with MALDI-TOF-MS analysis

The oleic acid‐functionalized magnetite nanoparticles (OA‐Fe₃O₄) with mean diameter of about 15 nm were synthesized through a low‐cost, one‐pot method and were designed as hydrophobic probes to realize the convenient, efficient and fast concentration of low‐concentration peptides followed by MALDI‐TOF‐MS analysis. The capability of OA‐Fe₃O₄ nanoparticles in concentration of low‐abundance peptides from simple and complex solutions were evaluated by comparing them with a sort of C8‐modified magnetic microspheres. Samples of standard peptide solution, protein digest solution and human serum were introduced in the evaluating process, and the OA‐Fe₃O₄ nanoparticles exhibited good surface affinity toward low‐concentration peptides     

Cytotoxicity,permeability, and inflammation of metal oxide nanoparticles in human cardiac microvascular endothelial cells

Wide applications and extreme potential of metal oxide nanoparticles (NPs) increase occupational and public exposure and may yield extraordinary hazards for human health. Exposure to NPs has a risk for dysfunction of the vascular endothelial cells. The objective of this study was to assess the cytotoxicity of six metal oxide NPs to human cardiac microvascular endothelial cells (HCMECs) in vitro. Metal oxide NPs used in this study included zinc oxide (ZnO), iron(III) oxide (Fe₂O₃), iron(II,III) oxide (Fe₃O₄), magnesium oxide (MgO), aluminum oxide (Al₂O₃), and copper(II) oxide (CuO). The cell viability, membrane leakage of lactate dehydrogenase, intracellular reactive oxygen species, permeability of plasma membrane, and expression of inflammatory markers vascular cell adhesion molecule-1, intercellular adhesion molecule-1, macrophage cationic peptide-1, and interleukin-8 in HCMECs were assessed under controlled and exposed conditions (12–24 h and 0.001–100 μg/ml of exposure). The results indicated that Fe₂O₃, Fe₃O₄, and Al₂O₃ NPs did not have significant effects on cytotoxicity, permeability, and inflammation response in HCMECs at any of the concentrations tested. ZnO, CuO, and MgO NPs produced the cytotoxicity at the concentration-dependent and time-dependent manner, and elicited the permeability and inflammation response in HCMECs. These results demonstrated that cytotoxicity, permeability, and inflammation in vascular endothelial cells following exposure to metal oxide nanoparticles depended on particle composition, concentration, and exposure time.     

In vitro spectroscopic investigation of groove binding interaction of Fe3O4@CaAl-LDH@L-Dopa with calf thymus DNA

Abstract

The principal goal of this study is to evaluate the interaction of Fe₃O₄@CaAl-LDH@L-Dopa and Fe₃O₄@CaAl-LDH nanoparticles with calf thymus DNA. The magnetic nanoparticles were previously prepared by a chemical co-precipitation method, and the surface of the Fe₃O₄ nanoparticles was coated with CaAl layered double hydroxides. The antiparkinsonian drug “L-Dopa” was carried by this core–shell nanostructure to achieve the drug delivery system with suitable properties for biological applications. Also, the interaction of Fe₃O₄@CaAl-LDH@L-Dopa and Fe₃O₄@CaAl-LDH nanoparticles with CT-DNA was studied using, UV–Visible spectroscopy, viscosity, circular dichroism (CD), and fluorescence spectroscopy techniques. The results of investigations demonstrated that Fe₃O₄@CaAl-LDH@L-Dopa and Fe₃O₄@CaAl-LDH nanoparticles have interacted via minor groove binding and intercalated to CT-DNA, respectively.     

Magnetic core/shell Fe3O4/Au nanoparticles for studies of quinolones binding to protein by fluorescence spectroscopy

Magnetic core/shell Fe₃O₄/Au nanoparticles were used in the determination of drug binding to bovine serum albumin (BSA) using a fluorescence spectroscopic method. The binding constants and number of binding sites for protein with drugs were calculated using the Scatchard equation. Because of their superparamagnetic and biocompatible characteristics, magnetic core/shell Fe₃O₄/Au nanoparticles served as carrier proteins for fixing proteins. After binding of the protein to a drug, the magnetic core/shell Fe₃O₄/Au nanoparticles–protein–drug complex was separated from the free drug using an applied magnetic field. The free drug concentration was obtained directly by fluorescence spectrometry and the proteins did not influence the drug determination. So, the achieved number of binding sites should be reliable. The binding constant and site number for ciprofloxacin (CPFX) binding to BSA were 2.055 × 10⁵ L/mol and 31.7, and the corresponding values for norfloxacin (NOR) binding to BSA were 1.383 × 10⁵ L/mol and 38.8. Based on the achieved results, a suitable method was proposed for the determination of binding constants and the site number for molecular interactions. The method was especially suitable for studies on the interactions of serum albumin with the active ingredients of Chinese medicine. Copyright © 2015 John Wiley & Sons, Ltd.     

Biodesulfurization of Dibenzothiophene by Microbial Cells Coated with Magnetite Nanoparticles

Microbial cells of Pseudomonas delafieldii were coated with magnetic Fe₃O₄ nanoparticles and then immobilized by external application of a magnetic field. Magnetic Fe₃O₄ nanoparticles were synthesized by a coprecipitation method followed by modification with ammonium oleate. The surface-modified Fe₃O₄ nanoparticles were monodispersed in an aqueous solution and did not precipitate in over 18 months. Using transmission electron microscopy (TEM), the average size of the magnetic particles was found to be in the range from 10 to 15 nm. TEM cross section analysis of the cells showed further that the Fe₃O₄ nanoparticles were for the most part strongly absorbed by the surfaces of the cells and coated the cells. The coated cells had distinct superparamagnetic properties. The magnetization (δ_s) was 8.39 emu · g⁻¹. The coated cells not only had the same desulfurizing activity as free cells but could also be reused more than five times. Compared to cells immobilized on Celite, the cells coated with Fe₃O₄ nanoparticles had greater desulfurizing activity and operational stability.     

DNA binding and cytotoxicity studies of magnetic nanofluid containing antiviral drug oseltamivir

In this work, the possibility of preparing a nanoparticle with improved treatment properties was investigated. In this regard, synthesis, characterization, in vitro cytotoxicity and DNA binding of Fe₃O₄@oleate/oseltamivir magnetic nanoparticles (MNPs) were investigated. Fe₃O₄ nanoparticles were synthesized via chemical co-precipitation and coated by oleate bilayers. Then, Fe₃O₄@OA MNPs were functionalized with an antiviral drug (oseltamivir), for better biological applications. The MNPs were subsequently characterized by zeta sizer and Zeta potential measurements, Fourier transform infrared (FT-IR) spectroscopy, vibrating sample magnetometer (VSM) and transmission electron microscopy (TEM) analyses. The TEM image demonstrated that average sizes of Fe₃O₄@OA/oseltamivir MNPs were about 8?nm. The in vitro cytotoxicity of Fe₃O₄@OA/oseltamivir MNPs was studied against cancer cell lines (MCF-7 and MDA-MB-231) and compared with oseltamivir drug. The results illustrated that Fe₃O₄@OA/oseltamivir magnetic nanoparticles have better antiproliferative effects on the mentioned cell lines as compared with oseltamivir. Also, in vitro DNA binding studies were done by UV–Vis, circular dichroism, and Fluorescence spectroscopy. The results indicated that Fe₃O₄@OA/oseltamivir MNPs bound to DNA via groove binding. Moreover, this magnetic nanofluid has potential for magnetic hyperthermia therapy due to magnetic core of its nanoparticles.

Communicated by Ramaswamy H. Sarma     

Biomolecule-functionalized magnetic nanoparticles for flow-through quartz crystal microbalance immunoassay of aflatoxin B<Subscript>1</Subscript>

A flow-through quartz crystal microbalance (QCM) immunoassay method has been developed based on aflatoxin B₁ antibody (anti-AFB₁)-functionalized magnetic core-shell Fe₃O₄/SiO₂ composite nanoparticles (bionanoparticles) in this study. To construct such an assay protocol, anti-AFB₁, as a model protein, was initially covalently immobilized onto the Fe₃O₄/SiO₂ surface, and then the functionalized nanoparticles were attached to the surface of the QCM probe with an external magnet. The binding of target molecules onto the immobilized antibodies decreased the sensor’s resonant frequency, and the frequency shift was proportional to the AFB₁ concentration in the range of 0.3–7.0 ng/ml. The regeneration of the developed immunosensor was carried out via attaching or detaching the external magnet from the detection cell. In addition, the selectivity, reproducibility, and stability of the proposed immunoassay system were acceptable. Compared with the conventional ELISAs, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay method could be further developed for the immobilization of other antigens or biocompounds.     

Novel synthesis of mannosamine conjugated magnetic nanoparticles for purification and stabilization of human lysosomal β-mannosidase

Human β-mannosidase (MANB) was purified to homogeneity directly from lysosomes by using mannosamine conjugated magnetic (Fe₃O₄) nanoparticles, DE-52 cellulose, and sephadex G-200 chromatography. Fe₃O₄ nanoparticles were synthesized and utilized ammonia to attach the amino group on the nanoparticles. The particles were covalently attached with D-mannosamine by cross linker glutaraldehyde and confirmed by FTIR spectroscopy. In FTIR analysis, the peaks appeared at 2,356.6 cm⁻¹ for −N = CH linkage and at 3,378.4 cm⁻¹, 3,664.9 cm⁻¹ for −OH groups confirmed the conjugation of D-mannosamine with Fe₃O₄ nanoparticles. Results showed a single band of 97 kDa of purified MANB in SDS-PAGE. The isoelectric point was 4.5 and the K_m and Vmax values were 2.51 mM and 0.315 μM/min/mg, respectively. The purification fold was 329 with 68% yield. The optimal activity was at pH 5.0 and 75% activity was stable in 20% glycerol at 4°C. The enzyme activity was inhibited by Ni²⁺, Zn²⁺, Cd²⁺, Cu²⁺, Mo²⁺, Ag⁺¹, iodoacetate, SDS, DMF, DMSO, ethanol, and acetone; slightly reduced by Pb²⁺, Co²⁺, EDTA, DTT, and β-mercaptoethanol. The activity was not affected by Mg²⁺, Mn²⁺, Sn²⁺, Ca²⁺, Fe³⁺, PMSF, Triton X-100, D-mannosamine, D-mannose, D-mannitol, D-glucose, and D-fructose. The homogeneity of MANB enzyme was further confirmed by 2D-PAGE and immunoblot. This is the first novel report of conjugation of D-mannosamine with Fe₃O₄ nanoparticles for purification of human MANB enzyme.     

The function of chitosan/agarose biopolymer on Fe2O3 nanoparticles and evaluation of their effects on MCF-7 breast cancer cell line and expression of BCL2 and BAX genes

In recent decades, magnetic nanoparticles modified with biocompatible polymers have been recognized as a suitable tool for treating breast cancer. The aim of this research was to evaluate the function of chitosan/agarose-functionalized Fe₂O₃ nanoparticles on the MCF-7 breast cancer cell line and the expression of BCL2 and BAX genes. Free Fe₂O₃ nanoparticles were prepared by hydrothermal method. FTIR, XRD, SEM, DLS, VSM, and zeta potential analyses determined the size and morphological characteristics of the synthesized nanoparticles. The effect of Fe₂O₃ free nanoparticles and formulated Fe₂O₃ nanoparticles on induction of apoptosis was studied by double-dye Annexin V-FITC and PI. Also, the gene expression results using the PCR method displayed that Fe₂O₃ formulated nanoparticles induced BAX apoptosis by increasing the anti-apoptotic gene expression and decreasing the expression of pro-apoptotic gene BCL2, so the cell progresses to planned cell death. In addition, the results showed that the BAX/BCL2 ratio decreased significantly after treatment of MCF-7 cells with free Fe₂O₃ nanoparticles, and the BAX/BCL2 ratio for Fe₂O₃ formulated nanoparticles increased significantly. Also, to evaluate cell migration, the scratch test was performed, which showed a decrease in motility of MCF-7 cancer cells treated with Fe₂O₃ nanoparticles formulated with chitosan/agarose at concentrations of 10, 50, 100, and 200 μg/ml.     

Tailoring size and structural distortion of Fe3O4 nanoparticles for the purification of contaminated water

Fe₃O₄ magnetic nanoparticles with different particle sizes were synthesized using two methods, i.e., a co-precipitation process and a polyol process, respectively. The atomic pair distribution analyses from the high-energy X-ray scattering data and TEM observations show that the two kinds of nanoparticles have different sizes and structural distortions. An average particle size of 6–8 nm with a narrow size distribution was observed for the nanoparticles prepared with the co-precipitation method. Magnetic measurements show that those particles are in ferromagnetic state with a saturation magnetization of 74.3 emu g⁻¹. For the particles synthesized with the polyol process, a mean diameter of 18–35 nm was observed with a saturation magnetization of 78.2 emu g⁻¹. Although both kinds of nanoparticles are well crystallized, an obviously higher structural distortion is evidenced for the co-precipitation processed nanoparticles. The synthesized Fe₃O₄ particles with different mean particle size were used for treating the wastewater contaminated with the metal ions, such as Ni(II), Cu(II), Cd(II) and Cr(VI). It is found that the adsorption capacity of Fe₃O₄ particles increased with decreasing the particle size or increasing the surface area. While the particle size was decreased to 8 nm, the Fe₃O₄ particles can absorb almost all of the above-mentioned metal ions in the contaminated water with the adsorption capacity of 34.93 mg/g, which is ∼7 times higher than that using the coarse particles. We attribute the extremely high adsorption capacity to the highly-distorted surface.     

Enhanced biodesulfurization by magnetic immobilized <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> LSSE8-1-vgb assembled with nano-γ-Al<Subscript>2</Subscript>O<Subscript>3</Subscript>

Biodesulfurization activity can be enhanced by assembling nano-γ-Al₂O₃ particles on the magnetic immobilized Rhodococcus erythropolis LSSE8-1-vgb. The cells can be collected and reused conveniently by an external magnetic field. Firstly, cells were magnetic immobilized by coating with Fe₃O₄ nano-particles. The optimal ratio of cells to magnetic Fe₃O₄ nano-particles was determined to be 50:1 (g/g). Then nano-γ-Al₂O₃ adsorbents were assembled onto the cells to enhance the desulfurization activity. The nano-γ-Al₂O₃ adsorbent had the largest pore volume as well as specific surface area, and the strongest electrostatics interaction with microbial cell, and cells assembled with this nano-adsorbent performed the highest desulfurization activity. The activity of magnetic immobilized cells assembled with adsorbents was tested in desulfurization of model oil. The desulfurization rate was raised by nearly 20% when the amount ratio of magnetic particles to adsorbents was 1:5 (g/g). These cells can be reused. The activity decreased less than 10% through out three desulfurization-activation-reuse recycles.     

Transport of Intranasally Instilled Fine Fe2O3 Particles into the Brain: Micro-distribution, Chemical States, and Histopathological Observation

It has been demonstrated that inhaled fine (d < 2.5 μm) and ultrafine (d < 100 nm) particles produce more severe toxicity than coarse particles. Some recent data support the concept that the central nervous system (CNS) may be a target for the inhaled fine particulates. This work describes initial observation of the transport of intranasally instilled fine ferric oxide (Fe₂O₃) particles in animal brain. The iron micro-distribution and chemical state in the mice olfactory bulb and brain stem on day 14 after intranasal instillation of fine Fe₂O₃ particle (280 ± 80 nm) suspension at a single dose of 40 mg/kg body weight were analyzed by synchrotron radiation x-ray fluorescence and x-ray absorption near-edge structure (XANES). The micro-distribution map of iron in the olfactory bulb and brain stem shows an obvious increase of Fe contents in the olfactory nerve and the trigeminus of brain stem, suggesting that Fe₂O₃ particles were possibly transported via uptake by sensory nerve endings of the olfactory nerve and trigeminus. The XANES results indicate that the ratios of Fe (III)/Fe (II) were increased in the olfactory bulb and brain stem. The further histopathological observation showed that the neuron fatty degeneration occurred in the CA3 area of hippocampus. Such results imply an adverse impact of inhalation of fine Fe₂O₃ particles on CNS.     

Glycosylation of various flavonoids by recombinant oleandomycin glycosyltransferase from <Emphasis Type="Italic">Streptomyces antibioticus</Emphasis> in batch and repeated batch modes

An oleandomycin glycosyltransferase (OleD GT) gene from Streptomyces antibioticus was functionally expressed in Escherichia coli BL21 (DE3) with various molecular chaperones. The purified recombinant OleD GT catalyzed glycosylation of various flavonoids: apigenin, chrysin, daidzein, genistein, kaempferol, luteolin, 4-methylumbelliferone, naringenin, quercetin and resveratrol with UDP–glucose. 4.6 μg OleD GT was readily immobilized onto 1 mg hybrid nanoparticles of Fe₃O₄/silica/NiO on the basis of the affinity between His-tag and NiO nanoparticles with retention of 90% activity. In batch reaction, more than 90% naringenin (20 μM) was converted to its glycoside in 5 h. The immobilized OleD GT was efficiently reused for seven times whilst maintaining >60% of the residual activity in repeated glycosylation of naringenin.     

In situ magnetic separation and immobilization of dibenzothiophene-desulfurizing bacteria

In situ cell separation and immobilization of bacterial cells for biodesulfurization were developed by using superparamagnetic Fe₃O₄ nanoparticles (NPs). The Fe₃O₄ NPs were synthesized by coprecipitation followed by modification with ammonium oleate. The surface-modified NPs were monodispersed and the particle size was about 13 nm with 50.8 emu/g saturation magnetization. After adding the magnetic fluids to the culture broth, Rhodococcus erythropolis LSSE8-1 cells were immobilized by adsorption and then separated with an externally magnetic field. The maximum amount of cell mass adsorbed was about 530 g dry cell weight/g particles to LSSE8-1 cells. Analysis showed that the nanoparticles were strongly absorbed to the surface and coated the cells. Compared to free cells, the coated cells not only had the same desulfurizing activity but could also be easily separated from fermentation broth by magnetic force. Based on the adsorption isotherms and Zeta potential analysis, it was believed that oleate-modified Fe₃O₄ NPs adsorbed bacterial cells mainly because of the nano-size effect and hydrophobic interaction.     

In situ preparation of magnetic Fe3O4-chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution

A new and simple method has been proposed to prepare magnetic Fe₃O₄-chitosan (CS) nanoparticles by cross-linking with sodium tripolyphosphate (TPP), precipitation with NaOH and oxidation with O₂ in hydrochloric acid aqueous phase containing CS and Fe(OH)₂, and these magnetic CS nanoparticles were used to immobilize lipase. The effects on the sequence of adding NaOH and TPP, the reaction temperature, and the ratio of CS/Fe(OH)₂ were studied. TEM showed that the diameter of composite nanoparticles was about 80 nm, and that the magnetic Fe₃O₄ nanoparticles with a diameter of 20 nm were evenly dispersed in the CS materials. Magnetic measurement revealed that the saturated magnetisation of the Fe₃O₄-CS nanoparticles could reach 35.54 emu/g. The adsorption capacity of lipase onto nanoparticles could reach 129 mg/g; and the maximal enzyme activity was 20.02 μmol min⁻¹ mg⁻¹ (protein), and activity retention was as high as 55.6% at a certain loading amount.     

巨噬细胞膜仿生纳米铁颗粒制备及多形性胶质母细胞瘤MRI成像的初步实验研究

摘要 目的：探讨巨噬细胞膜仿生的纳米铁颗粒（Fe3O4 NCs@MM）对多形性胶质母细胞瘤MRI成像的研究。方法：制备巨噬细胞膜仿生的纳米铁颗粒Fe₃O₄ NCs@MM,利用动态光散射（Dynamic Light Scattering,DLS）和透射电子显微镜（Transmission Electron Microscope,TEM）对其水合动力学粒径、表面电势和形态进行表征。采用SDS-聚丙烯酰胺凝胶电泳（sodium dodecyl sulphate-polyacrylamide gel electrophoresis,SDS-PAGE）评价巨噬细胞膜的完整包覆;紫外可见光谱测定巨噬细胞膜仿生的纳米铁颗粒抗蛋白吸附能力。通过MRI成像系统,分析了含不同浓度的Fe元素（0.1-1.6 mM）的Fe₃O₄ NCs@MM在GSH存在或不存在时的T₁弛豫效应。采用细胞增殖-毒性实验（Cell Counting Kit-8,CCK-8）,测定巨噬细胞膜仿生纳米铁颗粒处理肿瘤细胞24 h后的细胞活性。尾静脉注射巨噬细胞膜仿生纳米铁颗粒至原位胶质母细胞瘤模型中,观察成像效果。结果：巨噬细胞膜仿生的纳米铁颗粒Fe₃O₄ NCs@MM的水合动力学粒径和表面电势分别为 286.5±7.6 nm和-20.7±3.5 mV,且在水溶液中分布均匀,具有较好的单分散性。包覆巨噬细胞膜的纳米铁颗粒具备抗蛋白吸附的能力。MRI成像显示,制备的巨噬细胞膜仿生的纳米铁颗粒Fe₃O₄ NCs@MM为GSH响应型MRI对比剂,具有较好的T₁-加权磁共振成像效果,在尾静脉注射巨噬细胞膜的纳米铁颗粒0.5 h后,肿瘤部位的信号可见增强。结论：巨噬细胞膜仿生的纳米铁颗粒Fe₃O₄ NCs@MM可实现多形性胶质母细胞瘤的MRI成像。     

Extracellular biosynthesis of iron oxide nanoparticles by Bacillus subtilis strains isolated from rhizosphere soil

The biological synthesis of nanoparticles is emerging as a potential method for nanoparticle synthesis due to its non-toxicity and simplicity. We report the ability of Bacillus subtilis strains isolated from rhizosphere soil to produce iron oxide nanoparticles. B. subtilis strains having the potential for the extracellular biosynthesis of Fe₃O₄nanoparticles were isolated from rhizosphere soil, identified and characterized. A bactericidal protein subtilin was isolated from all the isolates of B. subtilis, which is a characteristic for the species. The isolated subtilin was tested against the bacterial strain, E. coli. The supernatant of the bacterial culture was used for the synthesis of Fe₃O₄ nanoparticles. The formation of nanoparticles was assessed by using UV-Visible spectrophotometer. FTIR and SEM analysis were used in order to confirm the formation and size of the nanoparticles. Further, the effect of incubation time, pH, and temperature on the formation of Fe₃O₄ nanoparticles was studied. The successful synthesis of stabilized Fe₃O₄ nanoparticles, which was capped by the organic group, indicates the applicability of the isolated B. subtilis strain for the bulk synthesis of iron oxide nanoparticles.     

Conjugation of α-chymotrypsin on a polymeric hydrophilic nanolayer covering magnetic nanoparticles

Magnetic nanoparticles, covered by a polymeric hydrophilic nanolayer containing reactive amino groups, were obtained via Hoffman degradation of the polyacrylamide-coated Fe₃O₄ nanoparticles synthesized by photochemical in situ polymerization, and then conjugated the model enzyme––α-chymotrypsin (CT) by use of EDC· HCl and NHS at room temperatures. The mechanism of photochemical in situ polymerization was briefly proposed in this paper. Superparamagnetic properties were retained for Fe₃O₄ after enzyme immobilization while slightly reducing the value of saturation magnetization. Crystalline structure of Fe₃O₄ after CT immobilization was consistent with that of the freshly prepared Fe₃O₄ by X-ray diffraction (XRD) analysis. The binding capacity was 69 and 61 mg enzyme/g nanogel determined by thermogravimetric (TG) analysis and by standard BCA protein assay, respectively. Specific activity of the immobilized CT was 0.93 U/(mg min), only 59.3% as that of free CT. Thermal stability of CT was improved after being bound to the amine-functionalized magnetic nanogel.