Synthesis of polyethylenimine modified Fe3O4 nanoparticles with immobilized Cu2+ for highly efficient proteins adsorption

Colloids and Surfaces A: Physicochem. Eng. Aspects 443 (2014) 552–559

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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa

Synthesis of polyethylenimine modified Fe3 O4 nanoparticles with immobilized Cu2+ for highly efficient proteins adsorption Tingting Xia a , Yueping Guan a,∗ , Mingzhu Yang a , Wubin Xiong a , Ning Wang b , Shen Zhao a , Chen Guo b a

School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China Laboratory of Separation Science and Engineering, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China b

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• PEI was modified on the Fe3 O4 nanoparticles using bifunctional GA as an intermediate. • The concentration of NH2 on the NPs increased from 0.2 mmol/g to 0.53 mmol/g. • The Fe3 O4 –PEI–Cu2+ NPs shows high adsorption capacity for BHb (6000 mg/g).

a r t i c l e

i n f o

Article history: Received 16 July 2013 Received in revised form 25 November 2013 Accepted 7 December 2013 Available online 22 December 2013 Keywords: Fe3 O4 –PEI NPs Protein adsorption BHb BSA Adsorption capacity Selectivity

a b s t r a c t We report the modification of amine-functioned Fe3 O4 nanoparticles (provided by the Laboratory of BioMedical Materials of University of Science and Technology Beijing) with polyethylenimine (PEI) using glutaraldehyde (GA) as a linker to increase the concentration of primary amine groups on the surface. The resultant Fe3 O4 –PEI NPs with immobilized iminodiacetic acid (IDA)–Cu2+ groups were applied to investigate the adsorption capacity and selectivity of model proteins: bovine hemoglobin (BHb) and bovine serum albumin (BSA). The Fe3 O4 –PEI NPs were characterized by transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), particle analyzer, attenuated total reflection-Fourier transform infrared spectroscopy (ATR–FTIR), ninhydrin colorimetry and thermogravimetric analysis (TGA). The results showed that the well dispersed Fe3 O4 –PEI NPs had an average size of 172.6 nm and were superparamagnetic with saturation magnetization of 71.11 emu/g. The characteristic peaks of PEI appeared in the ATR–FTIR spectra of Fe3 O4 –PEI NPs. The study of zeta potential measurement presented that the isoelectric points of the Fe3 O4 –PEI NPs increased to 10.5. Ninhydrin colorimetry analysis of primary amine groups showed that the concentration of primary amine groups on the surface of Fe3 O4 –PEI NPs increased to 0.45 mmol/g and that of TGA is 0.53 mmol/g. These results showed that PEI had been successfully modified on the surface of amine-functioned Fe3 O4 NPs and the concentration of primary amine groups was significantly increased. The analysis of protein adsorption indicated that the adsorption capacity of BHb was up to about 6000 mg/g and that of BSA was only about 2000 mg/g, which was about one third of that of BHb. The research on the selectivity of protein adsorption was conducted by HPLC. The result showed that the adsorption capacity of BHb was 2.5 times as high as that of BSA, which further proved Fe3 O4 –PEI–Cu2+ NPs shows specific adsorption for proteins. These properties suggested the potential of the Fe3 O4 –PEI–Cu2+ NPs as protein adsorbent. © 2013 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +86 10 62333945. E-mail address: [email protected] (Y. Guan). 0927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2013.12.026

T. Xia et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 443 (2014) 552–559

1. Introduction Recently, magnetic separation has attracted considerable attention for their increasing applications in biotechnology and biomedicine, especially in the separation of proteins due to ongoing need of purified proteins both in terms of diagnostics and therapeutics [1–5]. The basic principle of magnetic separation is simply to use magnetic particles modified with affinity ligands already utilized in chromatography [6] to be conjugated with proteins and isolated under the action of an external magnetic field. Fe3 O4 particles are most widely used as magnetic supports and endow magnetic separations with advantages of speed, easy use and affordability [7]. Magnetic separation has obtained some achievements in protein purification [8–12]. However, defects of magnetic adsorbents limit this method to analytical or laboratory scales, such as macroporous magnetic particles with relatively large specific surface areas that have big diffusion resistance within the particles and are easier to be “plugged” with biological foulants in their applications [13], or nonporous magnetic particles without sufficient active specific affinity ligands to provide linkage sites for protein adsorption [14]. It seems that nonporous magnetic particles with big concentration of functional groups can adsorb proteins efficiently. Therefore, it is of great significance to select nano-sized nonporous magnetic particles as raw material to be modified with suitable polymer in order to obtain enough reactive functional groups on the surface. PEI is a water-soluble cationic polymer with high density of primary, secondary and tertiary amine functional groups in each polymer chain (Scheme 1). As an ideal candidate for grafting polymer, PEI has been successfully applied both in terms of biomedicine [15–17] and environmental remediation [18,19]. The effectiveness of PEI in these applications should owe to its amine-rich structure, for instance, branched PEI (MW = 10,000 g/mol) has 81 primary amine groups per molecule. This amine-rich polymer will determine the surface charge and the number of available functional groups on the surface of the magnetic particles. Moreover, it is desired to enhance the dispersibility of magnetic particles by the electrostatic repulsive force and steric hindrance of the PEI [20]. It is also crucial to design an ideal ligand to guarantee the specific adsorption of target proteins by modified magnetic particles. Immobilized metal ions as pseudo affinity ligands have been of much interest since it was first introduced [21]. Such as Deniz Türkmen chelated Cu2+ on the PEI modified magnetic poly(2-hydroxyethylmethacrylate) (mPHEMA) beads to adsorb cytochrome c (cyt c) [22], Zheng Yeyou synthesized a novel IMAC material, zirconium-phosphate (Zr4+ -PO3 )-modified magnetic Fe3 O4 /GMA-co-EDMA (core/shell) (Zr4+ -Fe3 O4 @polymer) microspheres for the selective capture of phosphopeptides from protein tryptic digests[23] and so on. This kind immobilized metal affinity (IMA) separation is based on the interaction between chelated metal ions (Co2+ , Zn2+ , Ni2+ or Cu2+ ) and the thiol group cysteine, the indoyl group of tryptophan or especially the imidazole group of histidine of proteins [24]. Compared with classical bio-ligands, such as Protein A [25], metal ions have relatively low specificity, but they have higher physical and chemical stability, considerable bonding capacity and low cost, which is suitable for large-scale protein separation [26]. The iminodiacetic acid (IDA), the extensively used carboxymethylated amine, was selected as chelating ligand for Cu2+ . There exists two carboxyl per molecular of IDA, which strengthens the binding force between Cu2+ and magnetic particles compared with directly using primary amine groups modified on the magnetic particles to bond Cu2+ . Therefore, we selected nano-sized Fe3 O4 particles as inorganic magnetic core to be modified with branched PEI using bifunctional agent: glutaradehyde (GA) as reactive intermediate. The

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properties of the Fe3 O4 –PEI nanoparticles (NPs) were characterized by transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), attenuated total reflection-Fourier transform infrared spectroscopy (ATR–FTIR), particle analyzer, ninhydrin colorimetry and thermogravimetric analysis (TGA). The synthesized Fe3 O4 –PEI NPs immobilized Cu2+ using iminodiacetic acid (IDA) groups as chelating ligands, yielding Fe3 O4 –PEI–Cu2+ NPs to investigate the adsorption capacity and selectivity for model proteins: BHb and BSA. 2. Experimental 2.1. Materials Bare Fe3 O4 NPs (without primary amino groups on the surface) and amine-functioned Fe3 O4 NPs (the concentration of primary amine groups on the surface is 0.2 mmol/g and the size is 15 nm) were both provided by the Beijing GiGNano Biointerface Company. Glutaraldehyde (GA, 50% in water), sodium chloroacetate (CH2 ClCOONa) were supplied by Tian Jin Zhi Yuan Reagent Co., Ltd. (TianJin). Ethylenediamine (EDA), glacial acetic acid (HAC), cupric sulphate (CuSO4 ·5H2 O), sodium hydroxide (NaOH) and disodium ethylenediamine tetraacetate (EDTA) were purchased from Sinopharm Chemical Reagent Co., Ltd. (BeiJing). Polyethylenimine (PEI, branched, Mw = 10,000 g/mol), sodium borohydride (NaBH4 ) and tris (Hydroxymethyl)–aminomethane (Tris) were acquired from Aladdin (Shanghai). Bovine serum albumin (BSA) and bovine hemoglobin (BHb) were obtained from Roche (Switzerland). Deionized water and high purity water was provided by the Bio-Medical Materials laboratory of University of Science and Technology Beijing. All materials used by high performance liquid chromatography (HPLC) were of chromatographic grade and filtered with a 0.45 ␮m filter membrane purchased from Membrana Company (German). Other chemicals were of analytical grade. 2.2. Synthesis of PEI modified Fe3 O4 magnetic nanoparticles A given amount of amine-functioned Fe3 O4 NPs was added to 200 mL of a 10% (v/v) GA solution in water. The mixture was stirred at 25 ◦ C for 4 h in a constant temperature oscillator at 160 rpm, followed by washing with deionized water and a magnet many times until the excess GA were removed, yielding Fe3 O4 –GA NPs. Then these NPs were mixed with 300 ml of an aqueous solution of PEI (2.5%, w/v). The reaction continued conducting at 25 ◦ C for 4 h under vigorous stirring as described above, obtaining Fe3 O4 –GA–PEI NPs. Subsequently, 100 ml of EDA (80%, v/v) was added to react with unreacted aldehyde groups for 4 h under the above conditions. Whereafter, 15.4 g of NaBH4 (1 mol/L) was added to the reaction mixture in batch to change the obtained Schiff bases into more stable secondary amines. Finally, the synthesized NPs were washed as above to remove residual reagents and stored in deionized water. The procedures were illustrated in Scheme 1. 2.3. Immobilize IDA-Cu2+ on the surface of Fe3 O4 –PEI NPs The scheme of immobilizing IDA–Cu2+ on the surface of Fe3 O4 –PEI NPs is shown in Scheme 1. CH2 ClCOONa (8.0 g) was dissolved in 200 mL of deionized water, then a certain amount of Fe3 O4 –PEI NPs were added to the solution. The mixture reacted at 60 ◦ C for 8 h in a constant temperature oscillator at 200 rpm. NaOH solution was used to adjust the pH value to 11–12 throughout the whole process. The yielding products: Fe3 O4 –PEI–IDA NPs were washed with deionized water. Then these were mixed with 200 mL of CuSO4 ·5H2 O (0.4 mol/L) and the mixture was stirred vigorously at 30 ◦ C for 15 min. In the end, the Fe3 O4 –PEI–IDA–Cu2+ NPs

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Scheme 1. Schematic illustration for the synthesis of Fe3 O4 –PEI–IDA–Cu2+ NPs and their use for proteins adsorption.

were washed with deionized water for several times to remove the excess unbound Cu2+ . 2.4. Characterization of PEI modified Fe3 O4 magnetic nanoparticles The morphology and size of amine-functioned Fe3 O4 NPs were observed by transmission electron microscopy (TEM) at room temperature. The magnetic properties of the NPs were measured with a vibrating sample magnetometer (VSM) at 19 ◦ C. The particle size analysis of Fe3 O4 –PEI NPs and the zeta potential measurements at different pH were measured using the DelsaTM Nano C Particle Analyzer. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR–FTIR) analysis of amine-functioned Fe3 O4 NPs, Fe3 O4 –PEI NPs and PEI were taken on a T27-Hyperion-vector22 FTIR microscope (Bruker Corporation, Massachusetts, US) at room temperature. Comparing the amount of primary amine groups of the bare Fe3 O4 NPs, amine-functioned Fe3 O4 NPs, Fe3 O4 –GA NPs and Fe3 O4 –GA–PEI NPs was carried out via ninhydrin colorimetry. The reaction was conducted as follows: take the same quality (25 mg) of four kinds of NPs separately to react with ninhydrin solution (1 g/L, pH = 5.6) at 90 ◦ C (oil bath) for 15 min synchronously. Then the absorbance of the supernatants from the reaction mixture of NPs and ninhydrin was detected under identical conditions using ultraviolet spectrophotometer (UV-2100, Unico, America) at the wavelength of 570 nm. Quantitative analysis of organic compounds modified the Fe3 O4 NPs were performed using thermogravimetric analysis (TGA). They were recorded over the range of 25–800 ◦ C at the rate of 10 ◦ C/min. The density of Fe3 O4 –PEI–Cu2+ NPs dispersing in water and the chelated Cu2+ was measured by atomic absorption spectrophotometer (AAS, Shanghai Spectrum Instrument Co., Ltd.). 2.5. Proteins adsorption of Fe3 O4 –PEI–Cu2+ NPs BSA and BHb were selected as model proteins to explore the capacity and selectivity of Fe3 O4 –PEI–Cu2+ NPs for protein adsorption. The parallel experiments were carried as follows: according to the density of Fe3 O4 –PEI–Cu2+ NPs dispersing in

water (0.55 mg/mL), 1.0 mg of Fe3 O4 –PEI–Cu2+ NPs was taken out to mixed with 10 ml of BSA dissolved in 10 mM Tris–HAC buffer solution (pH = 8.1). The initial concentrations of BSA were in the range of 0.5–5.0 mg/mL. The experiment of BHb adsorption was conducted as above. Meanwhile, 6.0 mg of the NPs reacted with 10 ml of a binary mixture containing 2 mg/mL BHb and 2 mg/mL BSA dissolved in 10 mM Tris–HAC buffer solution (pH = 8.1). All the above experiments were conducted in an oscillator at 25 ◦ C for 10 min. After reaction, the NPs were magnetically separated and the supernatants were collected by high speed centrifuge at the speed of 8000 rpm for 30 min. According to the linear relationship between the concentration of single protein and the absorbance at 280 nm measured by UV-2100 spectrophotometer or the peak area measured by high performance liquid chromatography (HPLC, LC20A, Shimadzu, Japan), in the study of protein adsorption capacity, the initial and final concentration of single protein was measured by UV-2100 spectrophotometer and that of the binary mixture were detected by HPLC with Shim-pack WAX-2 column (Shimadzu, Japan). 3. Results and discussion 3.1. Synthesis and characterization of PEI modified Fe3 O4 magnetic nanoparticles Scheme 1 schematically shows the preparation process of PEI modified Fe3 O4 magnetic nanoparticles. GA as the reactive intermediate modified amine-functioned Fe3 O4 NPs with PEI through the reaction between aldehyde groups from GA and primary amino groups from amine-functioned Fe3 O4 NPs and PEI respectively. The added EDA reacted with the aldehyde groups that have not bonded with PEI. Generated imines which contained C N were changed into more stable secondary amines by NaBH4 . The added EDA and the positively charged PEI help to prevent the agglomeration of the synthesized NPs through electrostatic repulsion and steric hindrance [20]. TEM image and size distribution of the Fe3 O4 –PEI NPs are shown in Fig. 1a and b, respectively. It can be seen the well dispersed Fe3 O4 particles have a spherical shape with a mean diameter of 15 nm. The appearance of some aggregated or interconnected particles in the image could be due to the process of TEM sample preparation. It includes the air-drying of the moisture of the samples, which

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Fig. 2. Magnetic hysteresis loops of the amine-functioned Fe3 O4 NPs and Fe3 O4 –PEI NPs.

Fig. 1. TEM micrograph (a) and particle size distribution of Fe3 O4 –PEI NPs (b).

always leads to the aggregation or interconnection of the particles [27]. Because of PEI modified on Fe3 O4 NPs is amorphous, it can hardly been observed by TEM. So the TEM image actually demonstrates the morphology of the magnetic Fe3 O4 core. As shown in Fig. 1b, Y-axis of solid line is on the left. Y-axis of dotted line is on the right. And those were labeled by the arrows. When the size is bigger than 120 nm, the differential number increased, and reached to the peak at 141 nm, then decreased gradually to 0. When the size increased to 350 nm, the dotted line became flats. This suggests that the NPs have a narrow dimension distribution that is in the range of 120–350, the NPs of 141 nm accounted for the largest proportion of all the measured NPs and there hardly exist NPs bigger than 350 nm. The average size is 172.6 ± 47.1 nm, which is much smaller than that of other kinds of PEI modified Fe3 O4 particles, such as the PEI grafted Fe3 O4 @ mesoporous silica microsphere [28] and the PEI modified magnetic poly(2-hydroxyethylmethacrylate) (mPHEMA) beads [22]. The specific surface area of Fe3 O4 –PEI NPs increases to 7.0 m2 /g due to the nano size. Thus it can provide enough linkage sites for application. The typical magnetization curves applied magnetic field (M–H loops) of the amine-functioned Fe3 O4 NPs and Fe3 O4 –PEI NPs are shown in Fig. 2. The saturated magnetization of the aminefunctioned Fe3 O4 NPs and Fe3 O4 –PEI NPs is approximately 72.72 emu/g and 71.11 emu/g respectively and both behaved superparamagnetically. The saturated magnetization of Fe3 O4 –PEI NPs is slightly lower than that of amine-functioned Fe3 O4 NPs, which

indicates massive GA–PEI and little GA–EDA has covalently linked on the surface of amine-functioned Fe3 O4 NPs. It is much higher than that of magnetic polymer particles [29,30] and also higher than that of the similar PEI modified Fe3 O4 NPs [16,31]. This high saturated magnetization makes the NPs very susceptible to magnetic fields and easy to be separated from liquid phases with an ordinary permanent magnet. Moreover, the NPs with superparamagnetic property can redisperse rapidly when the magnetic field is removed, which plays a crucial role in biomedical application. The zeta potentials of the amine-functioned Fe3 O4 NPs, Fe3 O4 –GA NPs and Fe3 O4 –GA–PEI NPs in solutions were measured separately under different pH conditions adjusted using HCl or NaOH solution. As shown in Fig. 3, the zeta potential of Fe3 O4 NPs declined as the pH increased. The isoelectric point of amine-functioned Fe3 O4 NPs was 7.15. While that of Fe3 O4 –GA NPs decreased to 4.6. This is because GA reacted with the primary amine groups of amine-functioned Fe3 O4 NPs, yielding Fe3 O4 –GA NPs modified with aldehyde groups, which makes the NPs carry a negative surface charge at low pH. However, after PEI coated on the surface of the Fe3 O4 –GA NPs via reacting between aldehyde groups of Fe3 O4 –GA NPs and primary amine groups of PEI, the isoelectric point of Fe3 O4 –GA–PEI NPs significantly increased to 10.5, which was attributed to the protonation of imine groups on the NPs surface. The average pKa of PEI is reported to be 9.5 for the massive primary amine [32]. Accordingly, the Fe3 O4 –PEI NPs remained positively charged over a wide range of pH from 6.5 to 10.5. The results

Fig. 3. Zeta potentials of amine-functioned Fe3 O4 NPs (b), Fe3 O4 –GA NPs (a) and Fe3 O4 –GA–PEI NPs (c) as a function of pH.

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Fig. 5. The ATR–FTIR spectra of amine-functioned Fe3 O4 NPs (a), Fe3 O4 –PEI NPs (b) and PEI (c).

Fig. 4. The supernatants of reaction mixture of ninhydrin with bare Fe3 O4 NPs (A), amine-functioned Fe3 O4 NPs (B), Fe3 O4 –GA NPs (C) and Fe3 O4 –GA–PEI NPs (D) separately (a) and the absorbance of four kinds of supernatants (b).

suggest PEI has successfully covalently modified on the surface of original Fe3 O4 NPs. Ninhydrin solution can react with amino acid when heated together in weak acid condition and produce purplish blue end product, whose maximum absorption peak is at 570 nm when measured by UV-2100 spectrophotometer. The concentration of amino acid is directly proportional to the absorbance at 570 nm or the intensity of color. Detection of the concentration of primary amine groups on the surface of the NPs utilized this principle. The supernatants from the reaction between four kinds of Fe3 O4 NPs and ninhydrin solution and the corresponding absorbance of the supernatants were presented in Fig. 4a and b separately. As shown in Fig. 4a, the chromogenic extent of four kinds of supernatants is obviously different, which is corresponding to the data in Fig. 4b. We can see the color of the supernatants from aminefunctioned Fe3 O4 NP is light blue-violet and that of bare Fe3 O4 NPs without primary amino groups on the surface is almost colorless, which is corresponding to the result that the absorbance from amine-functioned Fe3 O4 NPs is much bigger than that from bare Fe3 O4 NPs. After GA covalently bonded on the amine-functioned Fe3 O4 NPs, the absorbance from Fe3 O4 –GA NPs becomes close to 0 and the color of the supernatant becomes colorless again. This indicates nearly all the primary amino groups on the surface of amine-functioned Fe3 O4 NPs have reacted with aldehyde groups of GA. The color of the supernatant from Fe3 O4 –PEI NPs is dark blue-violet and the absorbance from Fe3 O4 –PEI NPs is about twice as high as that from amine-functioned Fe3 O4 NPs, judging that the

concentration of primary amino groups on the NPs has greatly increased for the PEI modified on the surface of Fe3 O4 –GA NPs. According to the linear relationship between absorbance and concentrations of primary amino groups, the concentration of primary amino groups on the Fe3 O4 –PEI NPs is approximately 0.45 mmol/g, which is about 2.25 times as high as that of amine-functioned Fe3 O4 NPs. Fig. 5 presents the ATR–FTIR spectra of amine-functioned Fe3 O4 NPs (a), Fe3 O4 –PEI NPs (b) and PEI (c). Fig. 5(c) shows the characteristic absorption peaks of PEI at 3356 cm−1 (N H stretching vibration), 2936 cm−1 and 2810 cm−1 (C H stretching vibration), 1458 cm−1 (C H scissoring bending vibration), 1122 cm−1 and 1037 cm−1 (C N stretching vibration). The peaks appearing both in Fig. 5(a) and (b) are at 551 cm−1 corresponds to Fe O vibration of Fe3 O4 , 3400 cm−1 corresponds to N H stretching vibration, 1640 cm−1 and 1540 cm−1 correspond to N H bending vibration, 1320 cm−1 corresponds to C N stretching vibration. Those demonstrate the existence of Fe3 O4 and amino groups on the surface of NPs. It can be observed some new absorption peaks appeared in the spectrum of synthesized Fe3 O4 –PEI NPs compared with that of amine-functioned Fe3 O4 NPs. These bands are at 2948.02 cm−1 , 2827.20 cm−1 , 1472.90 cm−1 and 1064.68 cm−1 , which correspond to C H stretching vibration, C H stretching vibration, C H scissoring bending vibration and C N stretching vibration respectively in the PEI structure shown in Fig. 5c. Hence, these observations provide evidence that PEI has been successfully immobilized on the surface of amine-functioned Fe3 O4 NPs. The TGA analyses of amine-functioned Fe3 O4 NPs and Fe3 O4 –PEI NPs are shown in Fig. 6. It shows that the weight-loss of aminefunctioned Fe3 O4 NPs in the range of 100 ◦ C to 625 ◦ C is 4.073 wt%, resulting from the decomposition of organic compounds modified on the surface of amine-functioned Fe3 O4 NPs and the oxidation of Fe3 O4 NPs. In the same region, the weight-loss of Fe3 O4 –PEI NPs increases to 10.56 wt%. This could be due to GA–PEI–NaBH4 and little GA–EDA–NaBH4 modified on the surface of amine-functioned Fe3 O4 NPs. By subtracting the weight-loss of amine-functioned Fe3 O4 NPs and neglecting the weight of the molecular with low molecular-weight (GA, EDA, NaBH4 ), we can estimate the weight of PEI modified on the surface of amine-functioned Fe3 O4 NPs accounts for 6.487 wt%. That is to say the modifying capability of PEI is about 6.487 ␮mol/g, which increases the concentration of primary amine groups on the NPs surface to 0.53 mmol/g. This is close to the result of ninhydrin colorimetry. The significantly improved concentration should owe to the amine-rich structure of PEI, which can provide much more linkage sites for protein adsorption.

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Fig. 6. Thermogravimetric Fe3 O4 –GA–PEI NPs.

curves

of

amine-functioned

Fe3 O4

NPs

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and

3.2. Protein adsorption of Fe3 O4 –PEI NPs with immobilized IDA-Cu2+ As shown in Scheme 1, the IDA group utilized as chelating ligand for Cu2+ has two carboxyl per molecular, which strengthens the binding force between Cu2+ and Fe3 O4 –PEI NPs. The adsorption of proteins relies on the great affinity between the imidazolyl group of surface-exposed histidine residues from BHb or BSA and immobilized Cu2+ . The proteins adsorption capacity was determined by the following Eq. (1): q=

(c0 − c)V m

(1)

where q is the density of adsorbed protein in equilibrium (mg/g), qmax is the maximum adsorption capacity (mg/g), c0 is the initial concentrations of protein (mg/mL), c is the equilibrium protein concentration (mg/mL), V is the volume of protein solution (mL), m is the weight of Fe3 O4 –PEI–Cu2+ NPs (g). The adsorption isotherms of BSA and BHb are presented in Fig. 7a. It is observed that the equilibrium adsorption capacity tends to a maximum with the increasing of equilibrium proteins concentration. The qmax of BHb and BSA is estimated at 6000 mg/g (0.093 mmol/g) and 2000 mg/g (0.031 mmol/g) respectively. According to the analysis of TGA, 1 mg of Fe3 O4 –PEI NPs has about 6.478 ␮mol of PEI. Assuming that all primary amine groups of PEI had bonded with IDA–Cu2+ and every chelated Cu2+ had adsorbed one molecule of protein, we can roughly calculate out the theoretical adsorption capacity of Fe3 O4 –PEI–Cu2+ NPs for BHb is 0.525 mmol/g. On the other hand, according to the results of AAS, the concentration of Cu2+ chelated on the surface of Fe3 O4 –PEI NPs is 0.22 mmol/g. This amount can make 1 g of the NPs absorb 0.22 mmol of BHb if all Cu2+ bonded with proteins. The results of the two aspects suggest that success rate of chelating Cu2+ is 41.90% and that of adsorbing BHb by prepared NPs is 42.28%. The result shows that the adsorption capacity of BHb is much higher than those of similar works, such as Z. Ma prepared micronsized magnetic particles with tentacle type polymer chains on the surface, whose adsorption capacity of BHb was only 1428.21 mg/g [29]. This significant increase of adsorption capacity is due to the small particle size (172.6 nm), which increases the specific surface area of particles to 7.0 m2 /g and massive linkage sites on the surface of Fe3 O4 –PEI NPs obtained by grafting more branched PEI. On the other hand, the smaller qmax of BSA indicates the selective adsorption of Fe3 O4 –PEI–Cu2+ NPs for proteins.

Fig. 7. Adsorption isotherms of BHb and BSA on Fe3 O4 –PEI–IDA–Cu2+ NPs (10 Mm Tris–HAC buffer solution, pH = 8.1) (a), the Langmuir fitting (b) and the Temkin fitting (c) of the adsorption data.

The Langmuir model could be described as follows: 1 Kd 1 = + q cqmax qmax

(2)

where q is the equilibrium adsorption capacity (mg/g), c is the equilibrium protein concentration in supernatants (mg/mL), qmax is the maximum adsorption capacity (mg/g) and Kd is the Langmuir adsorption constant (mg/mL). Fig. 7b shows the double reciprocal plots of BHb and BSA and the fitting curves of the experiment dates

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linkage sites on the surface of NPs. After chemical modification with IDA–Cu2+ groups to form magnetic adsorbents, the Fe3 O4 –PEI–Cu2+ NPs showed very high adsorption capacity for BHb (6000 mg/g) and little adsorption capacity for BSA (2000 mg/g). The adsorption mode of BSA accords with the Langmuir model because of the weak interaction between the contiguous molecules. The adsorbed BHb on NPs has strong interaction between the contiguous molecules and the adsorption model more accords with Temkin model than Langmuir model. The NPs also presented high selectivity for BHb in the binary mixture. Furthermore, this method is facile in operation, rapid, and low cost. With these advantages, the Fe3 O4 –PEI–Cu2+ NPs show great potential to be served as magnetic adsorbents to separate or purify biomolecules in large scale.

Fig. 8. HPLC chromatograms of the binary mixture (2 mg/mL BHb and 2 mg/mL BSA) before and after proteins adsorption by Fe3 O4 –PEI–IDA–Cu2+ NPs.

to the Langmuir equation. It indicates that BSA adsorption obeys the Langmuir adsorption model. While BHb adsorption does not. This could be due to the assumptions of Langmuir model that it has neglected the interaction between the contiguous molecules. While BSA and BHb as the biological macromolecules have much larger volume size compared with small molecules. The macromolecule first adsorbed on the NPs may form large steric resistance and interaction between the contiguous molecules, which will affect the adsorption of the subsequent macromolecules. According to the result that the adsorption capacity of BSA is much less than that of BHb, it can be inferred that interaction between the contiguous molecules of absorbed BSA is much weaker than that of adsorbed BHb. So the linear correlation coefficient (R2 ) of BSA fitting curve was 0.93, while that of BHb was only 0.61. The Temkin model is the non-ideal monolayer adsorption model considering the factors of heterogeneous surface and interaction between absorbed molecules. It could be described as follows: q = K ln c + K ln f

(3)

where q is the equilibrium adsorption capacity (mg/g), c is the equilibrium protein concentration in supernatants (mg/mL), K and f are the adsorption constants. Fig. 7c shows the Temkin fitting of these two kinds of proteins. The R2 of BSA fitting curve increased little. While the R2 of BHb increased to 0.81, which suggested that the Temkin model was more suitable for BHb adsorption for the existence of strong interaction between the absorbed BHb. The selectivity of proteins adsorption is further confirmed by the change of peak area belonging to BHb and BSA in HPLC chromatograms before and after proteins adsorption shown in Fig. 8. The peak area of BHb decreased much more after adsorbed by Fe3 O4 –PEI–Cu2+ NPs than that of BSA. According to Eq. (1), the amounts of BHb and BSA adsorbed from the binary mixture by the NPs are found to be 999.305 mg/g and 406.704 mg/g, respectively. That is because Cu2+ chelated on Fe3 O4 –PEI NPs has great affinity with the imidazolyl group of histidine residues [24,33]. There are 20 surface exposed histidine residues on BHb [34,35]. While, BSA has only 2 surfaces exposed histidine residues [36]. This promotes more adsorption of BHb. Overall, the Fe3 O4 –PEI–Cu2+ NPs show higher selective adsorption capacity for BHb than that for BSA in the binary mixture. 4. Conclusions Well-defined Fe3 O4 –PEI NPs were fabricated via a linker of GA. The magnetic particles with a mean size of 172.6 nm exhibited superparamagnetism and the high saturation magnetization (71.11 emu/g). Branched PEI facilitated improving the concentration of primary amine groups to 0.53 mmol/g, which provided more

Acknowledgments This work was financially supported by National Natural Science Foundation of China (51274035) and National Basic Research Program of China (2013CB632602).

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