Applied Surface Science 256 (2010) 4596–4600 Contents lists available at ScienceDirect Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc Immobilization of cholesterol oxidase to finely dispersed silica-coated maghemite nanoparticles based magnetic fluid Franja ˇ Sulek, ˇ Zeljko Knez ∗∗ , Maja Habulin ∗ University of Maribor, Faculty of Chemistry and Chemical Engineering, Laboratory for Separation Processes and Product Design, Smetanova 17, SI-2000 Maribor, Slovenia article info Article history: Received 28 September 2009 Received in revised form 16 February 2010 Accepted 17 February 2010 Available online 25 February 2010 Keywords: Magnetic nanoparticles Magnetic carrier Enzyme immobilization Cholesterol oxidase abstract In the recent years, the potential applicability of magnetic nanoparticles (MNPs) has witnessed a signifi- cant increase in interest towards the medical field, in particular, towards the usage of novel nanoparticles in diagnostics and disease treatment, respectively. In a present study, cholesterol oxidase (ChOx) was covalently immobilized to magnetic nanoparticles of maghemite (-Fe 2 O 3 ) and further functionalized by silica (SiO 2 ) and amino-silane molecules. The activity of the bound enzyme was retained up to 60%, respectively. The binding of cholesterol oxidase was confirmed using FT-IR spectrophotometer. SEM anal- ysis showed uniformly dispersed functional magnetic nanoparticles, which ranged in size from 22.5 to 50.8 nm, surrounded by amorphous silica. In this paper, the potential applications of chemically modified magnetic nanoparticles as carriers for cholesterol oxidase and other enzymes are discussed. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Stability of the enzyme is very often improved by immobiliza- tion [1]. So far, many organic and inorganic substances have been used as materials for the fabrication of a suitable carrier for a diverse range of biomolecules, for instance, the enzyme. Among them, magnetic particles on nanometer scale receive considerable attention because of their widely increased use in the immobi- lization procedures of proteins and enzymes. Moreover, with the rapid development of nanostructured materials, the genesis of magnetic nanoparticles with improved characteristics brings the following advantages: (a) a significantly higher specific surface area is obtained for the attachment of a larger amount of enzyme per unit mass of nanoparticles, (b) lower mass transfer resis- tance is expected, and (c) the magnetically labelled immobilized enzymes can be selectively separated from a reaction mixture by the application of an external magnetic field [2–4]. Due to magnetic separation, it is possible to achieve very high efficiency of separa- tion of many bioactive substances such as enzymes and proteins. Other applications of magnetic particles include immunoassays, Abbreviations: AEAPS, 3-(2-aminoethylamino)-propyl-dimethoxymethylsilane; ChOx, cholesterol oxidase; FT-IR, Fourier transform infrared spectroscopy; E, desig- nation for enzyme; MNPs, magnetic nanoparticles; PBS, phosphate buffer solution; BSA, bovine serum albumin. ∗ Corresponding author. Fax: +386 2 2527 462. ∗∗ Corresponding author. Fax: +386 2 2527 408. E-mail addresses: zeljko.knez@uni-mb.si ( ˇ Z. Knez), maja.habulin@uni-mb.si (M. Habulin). drug targeting, drug transporting, and biosensing. Furthermore, the application of magnetic nanoparticles expands towards the removal of toxic elements from industrial wastes, respectively [5]. Modified magnetic materials are nowadays well-known and have been investigated intensively due to their potential appli- cations in enzyme binding protocols [6]. Maghemite (-Fe 2 O 3 ) is one of the famous magnetic materials in common use. Due to its magnetic character, -Fe 2 O 3 nanoparticles can be attracted by a magnetic field and are easily separable in solution, thereby, enabling also separation of bioactive substances that are linked directly or indirectly to the surface of MNPs. For biological appli- cations, it is very important to conjugate MNPs with appropriate reactive molecules, such as amino groups. The MNPs used in the present study were surrounded by amorphous SiO 2 , i.e., Si–OH (silanol group) was located on the outer surface ready to be further modified. Surface modification with amino silane reactive groups is very common method for particle functionalization. Nevertheless, amino silane molecules that act as coupling agents are conjugate to the surface of magnetic nanoparticles after deposition of inorganic silica [7,8]. Adversely, direct attachment of amino silane molecules onto particle surface has also been already encountered [9]. High density of free amino groups (–NH 2 ) lying outwards the particle surface provides an excellent media for further chemical surface modification such as enzyme cross-linking with glutaraldehyde. Suitable functional coating of magnetic particles has many bene- fits. For instance, particle size is notably increased, resulted in the slight decrease of reactivity of the particles. Thus, in this fashion the problem of particle agglomeration could be thoroughly omitted. 0169-4332/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2010.02.055