Immobilization of albumin on magnetite nanoparticles Esra Maltas a, ⁎, Mustafa Ozmen a , Hasibe Cingilli Vural b , Salih Yildiz a , Mustafa Ersoz a a Department of Chemistry, Selcuk University, Konya 42075, Turkey b Department of Biology, Selcuk University, Konya 42075, Turkey abstract article info Article history: Received 3 May 2011 Accepted 18 July 2011 Available online 22 July 2011 Keywords: Magnetic materials APTES Immobilization Biomaterials SDS-PAGE The magnetite (Fe 3 O 4 ) nanoparticles were prepared by the co-precipitation of ferrous and ferric salts with NH 4 OH, and then modified with 3-aminopropyltriethoxysilane (APTES) by silanization reaction and subsequent reaction with glutaraldehyde (GA) to obtain functional groups on their surface. The influence of different terminated groups on protein binding was studied with bare and modified magnetite nanoparticles. Amine terminated magnetite nanoparticles were shown the highest binding ability for immobilization process compared to Fe 3 O 4 NPs and GA bonded NPs. This binding ability was shown by using sodium dodecyl polyacrylamide gel electrophoresis technique (SDS-PAGE). Albumin attached magnetite nanoparticles were also examined by Scanning Electron Microscopy (SEM). © 2011 Elsevier B.V. All rights reserved. 1. Introduction In recent years, separation and purification of target protein and elucidation of protein function are some of the major tasks facing researchers. Thus, development of tools to enhance protein studies is critical. Many tools have been developed to purify or separate individual proteins from biological matrices. One of the tools that are magnetite particles (microspheres, nanospheres and ferrofluids) is widely used in purification, separation and immobilization of protein and enzymes. They are also used in the biomedical field as a solid support for immunoassays, DNA sequencing, and cell analysis and magnetically controlled transport of anti-cancer drugs [1–5]. Iron oxides magnetite particles are a group of the paramagnetic nano- particles modified with various functional group such as epoxy, amine and aldehyde that give better results for immobilization or binding. The application for biomolecules immobilization mainly based on the solid-phase magnetic feature which has the advantages of quick, easy, and gentle separation of biological compounds using an external magnetic field gradient [7–12]. For taking advantage of magnetic properties, many multifunctional materials have been reported for use in bioapplications [13–15]. Recently, some magnetic bead-based materials have been developed for cancer markers via immunoassay studies like antibody–antigen interactions by electrochemical analysis as an alternative method to HPLC or western blotting system [16,17]. Magnetic particles are also used in recombinant DNA technology. The production of recombinant fusion proteins having appropriate affinity tags like GST (glutathione-S-transferase) or His-tag (Histi- dine) labeled proteins is expressed by the specific genes in different host cells. This affinity tags make fast and easy purification of the target proteins from the complex biological samples. Magnetic batch separations, employing magnetic affinity and ion exchange particles, have been shown to be very useful for protein separations [18]. In this paper, magnetic Fe 3 O 4 nanoparticles were prepared by the chemical co-precipitation of Fe(III) and Fe(II) ions. Then, the nano- particles were modified by 3-aminopropyltriethoxysilane (APTES) to introduce reactive groups onto the particles surface, and subsequently with GA was grafted onto modified nanoparticles by surface. The functionalized magnetic particles were used for protein binding. 2. Materials and methods Superparamagnetic magnetite nanoparticles were prepared via improved chemical co-precipitation method [6]. APTES and subse- quently GA modification on magnetite nanoparticles was prepared as before [21]. Prepared NPs were mixed with 2 mg/mL albumin in 1× PBS, pH 7.4 and thumbled overnight at 4 °C at a particle concentration range of 5–30 mg/mL. Supernatant was removed and kept at 4 °C for further protein analysis after BSA bounded Fe 3 O 4 particles separated magnetically. Albumin bounded particles were also washed with PBS and ethanol for chemical characterization. Protein concentration was determined using Bradford (1976) method [19] according to the manufacturer's instructions. Remaining protein after albumin binding on NPs was diluted to appropriate concentrations. 3 mL of Bradford reagent was mixed 100 μL of each mixture and absorbance was recorded at 595 nm. Unbounded protein concentration was calculated from standard curve equation of the BSA (y = 0.7725x). Samples were examined via SDS-PAGE as described by Laemmli [20]. Briefly, albumin immobilized particles were mixed with 3× SDS sample buffer and Materials Letters 65 (2011) 3499–3501 ⁎ Corresponding author. Tel.: +90 332 223 38 96; fax: +90 332 241 24 99. E-mail address: esramaltas@gmail.com (E. Maltas). 0167-577X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2011.07.045 Contents lists available at ScienceDirect Materials Letters journal homepage: www.elsevier.com/locate/matlet