Adsorption of bovine serum albumin on nanosized magnetic particles

https://doi.org/10.1016/j.jcis.2003.12.022Get rights and content

Abstract

Adsorption of bovine serum albumin (BSA) on nanosized magnetic particles (Fe3O4) was carried out in the presence of carbodiimide. The equilibrium and kinetics of the adsorption process were studied. Nanosized magnetic particles (Fe3O4) were prepared by the chemical precipitation method using Fe2+, Fe3+ salts, and ammonium hydroxide under a nitrogen atmosphere. Characterizations of magnetic particles were carried out using transmission electron microscopy (TEM) and a vibrating sample magnetometer (VSM). Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to confirm the attachment of BSA on magnetic particles. Effects of pH and salt concentrations were investigated on the adsorption process. The experimental results show that the adsorption of BSA on magnetic particles was affected greatly by the pH, while the effect of salt concentrations was insignificant at a low concentration range. The adsorption equilibrium isotherm was fitted well by the Langmuir model. The maximum adsorption of BSA on magnetic particles occurred at the isoelectric point of BSA. Adsorption kinetics was analyzed by a linear driving force mass-transfer model. BSA was desorbed from magnetic particles under alkaline conditions, which was confirmed by SDS–PAGE electrophoresis and FTIR results.

Introduction

Magnetic separation is a recent developing technology and mostly applied in the field of bioseparation. The principle of this method is to utilize magnetic particles to bind the target molecules via ligand to form a complex that can be separated from the bulk solution by magnetic field gradient. Its application includes enzyme immobilization [1], [2], cell sorting [3], [4], [5], protein adsorption and purification [6], [7], nucleic acid detachment [8], [9], and drug delivery [10]. Compared to conventional separation, the advantages of magnetic separation are attributed to its speed, accuracy, and simplicity.

Many published works focused on the synthesis of micrometer-sized polymer matrix containing magnetic particles and its application in the separation of protein with the aid of a specific ligand coating the surface of the particles [11], [12]. Only limited work has been published on the application of nanosized magnetic particles in the separation of proteins. Nanosized magnetic particles can produce larger specific surface areas and, therefore, may result in high adsorption capacity for proteins. Therefore, it may be useful to synthesize nanosized magnetic particles with large surface areas and utilize them as suitable carriers for the adsorption/desorption of protein.

Water-soluble carbodiimide is used to immobilize protein on carboxyl-terminated polymer. Chen and Chen [13] used carbodiimide for the immobilization of lysozyme on polymer, while Dilgimen et al. [14] utilized carbodiimide to conjugate BSA on polymer. Recently, carbodiimide was also used to activate the direct adsorption of BSA and alkaline phosphatase [15], [16] on magnetic particles. Yoon et al. [17] studied the separation of protein using submicrometer-sized magnetic particles without ligand and suggested that hydrogen bonding, hydrophobic interaction, and electrostatic repulsion may also promote the adsorption. Bergemann et al. [18] used nanosized (50 or 100 nm) magnetic particles coated with ionically susceptible ligand in bioseparation.

Desorption of protein from magnetic particles had been studied either by acid or alkaline washing. Safarik and Safarikova [19] used a 0.01 M HCl solution to desorp lysozyme from magnetic chitin. Khng et al. [11] found that an acetic acid solution containing salt was more efficient than HCl or glycine–HCl for the desorption of trypsin from magnetic particles. Honda et al. [20] used alkaline condition (pH 10–13) for the desorption of recombinant Escherichia coli from chitosan-conjugated magnetite. Yoshida and Kataoka [21] noted that the desorption of BSA from cross-linked chitosan could be done by an alkaline buffer solution.

Although there was some previous work carried out on protein adsorption/desorption on large sized magnetic particles, not much work has been published on protein adsorption/desorption on nanosized magnetic particles. In this work, the equilibrium and kinetics of BSA adsorption on nanosized magnetic particles were studied. The effect of pH and salt concentrations on adsorption was investigated in detail. Desorption of BSA was also studied.

Section snippets

Materials

Iron(II) chloride tetrahydrate (99%) was obtained from Fisher (USA). Iron(III) chloride hexahydrate (98%) was obtained from Nacalai Tesque (Japan). Ammonium hydroxide (25%) was purchased from Merck (USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, bovine serum albumin (BSA), and tris(hydroxymethyl)aminomethane were purchased from Sigma-Aldrich (USA). All the chemicals were used as received without further treatment. The water used in this work was Milli-Q ultrapure water.

Preparation of Fe3O4 magnetic particles

Characterization of magnetic particles

Size and morphology of magnetic particles were characterized by TEM. A typical TEM micrograph of magnetic particles is shown in Fig. 1. It shows that the size of magnetic particles is about 10 nm, which is comparable to the reported value 8.5 nm [23]. It is known that magnetic particles less than about 30 nm will exhibit superparamagnetism [7]. Therefore, the prepared magnetic particles (Fe3O4) have superparamagnetic properties and are expected to respond well to magnetic fields without any

Conclusions

Nanosized magnetic particles Fe3O4 were prepared by the chemical precipitation method and characterized by TEM and VSM for size and supermagnetic properties. Adsorption of BSA on magnetic particles was carried out in the presence of carbodiimide under different pH and salt concentrations. It has been found that pH has a great effect on BSA adsorption while the effect of salt at a lower concentration range was comparatively insignificant. The maximum adsorption of BSA on magnetic particles

Acknowledgements

This work was financially supported by the National University of Singapore Research Fund (R-279-000-085-112).

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