Surface modification of permalloy (Ni80Fe20) nanoparticles for biomedical applications
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- Qin, G.W., Darain, F., Wang, H. et al. J Nanopart Res (2011) 13: 45. doi:10.1007/s11051-010-0101-5
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We report a simple and novel method for surface biofunctionalization onto recently reported Ni80Fe20 permalloy nanoparticles (~71 nm) and the immobilization of a model protein, IgG from human serum. The strategy of protein immobilization involved attachment of histidine-tagged streptavidin to the Ni80Fe20 nanoparticles via a non-covalent ligand binding followed by biotinylated human IgG binding on the nanoparticle surface using the specific high affinity avidin–biotin interaction. The biofunctionalization of Ni80Fe20 permalloy nanoparticles was confirmed by Fourier Transform InfraRed (FTIR) spectroscopy and protein denaturing gel electrophoresis (lithium dodecyl sulfate-polyacrylamide gel electrophoresis, LDS-PAGE). This protocol for surface functionalization of the novel nanometer-sized Ni80Fe20 permalloy particles with biological molecules could open diverse applications in disease diagnostics and drug delivery.
KeywordsNi80Fe20 permalloyMagnetic nanoparticlesProtein immobilizationAvidin–biotin bridgeGel electrophoresisNanomedicine
Nanoparticles play a major role in the development of nanoscience and nanotechnology due to their high surface area to volume ratios, and unique physical and chemical properties (Tansil and Gao 2006). Magnetic nanoparticles functionalized with various biomolecules on their surfaces can be used as a highly effective platform for the development of diverse applications in medicine and biomedical sciences (Shinkai 2002; Lu et al. 2007; Couvreur and Puisieu 1993; Dave and Gao 2009; Veiseh et al. 2010; Chanana et al. 2009; Shubayev et al. 2009). Consequently, in recent years, magnetic nanomaterials have attracted considerable research interest due to their potential role in a wide range of bio-medical applications, such as drug delivery, bio-sensing, and disease detection, etc. (Ma et al. 2004; Nidumolu et al. 2006). In most applications, fine tuning is required for the magnetic nanomaterial properties, such as magnetic susceptibility, magnetization, and coercivity at ambient environment (Dave and Gao 2009). Recently, we have synthesized novel nanometer-sized Ni80Fe20 permalloy particles with tunable sizes (tens–hundreds nm in diameter) and narrow size distribution. These particles have low remnant magnetization and coercivity allowing dispersive behavior in aqueous solution, with minimal aggregation, and have very high magnetic susceptibility and sufficiently high saturation magnetization to trigger robust responses to external magnetic fields (Qin et al. 2009). However, functionalization of the novel Ni80Fe20 permalloy nanoparticles with biological molecules has not been studied. Therefore, it was important and interesting to bio-functionalize the permalloy nanoparticles in order to take full advantage of their superior magnetic properties and to provide room for new applications in nanobiotechnology.
Biological molecules can be immobilized on the surface of magnetic nanoparticles via covalent bonding using thiol chemistry (Sestier et al. 1998) and carbodiimide activation (Gan et al. 2008) by random amide bond formation, coating with silica (Jang and Lim 2010) or precious metals for further chemical functionalization, etc. (Li et al. 2010; Zhuo et al. 2009; Pham et al. 2008; Tang et al. 2006). However, these processes are time consuming and also associated with several steps or harsh chemical or physical treatments. On the other hand, it is well known that several metal ions exhibit affinity to poly-histidines with Nickel ions being the most recognizable among them. Lee et al. (2006) reported Ni/NiO core/shell nanoparticles for selective binding and magnetic separation of his-tagged proteins on the basis of the fact that NiO shell does have a high affinity for polyhistidine. The predominant Ni fraction in Ni80Fe20 permalloy nanoparticles is expected to form a NiO layer (Nam et al. 2004; Park et al. 2005; Johnston-Peck et al. 2009) while the metallic Ni80Fe20 permalloy remains as the core. Thus, the nano-sized Ni80Fe20 permalloy particles could potentially be combined with histidine-tagged biomolecules for further modification for biomedical applications.
In the present communication, a simple and facile method for biological functionalization of Ni80Fe20 magnetic nanoparticles has been studied without introducing time consuming processes or harsh chemical or physical treatments. To our knowledge, this is the first approach to report a biomolecule-functionalized Ni80Fe20 permalloy nanomaterial system. Streptavidin with a polyhistidine tail has been specifically attached via non-covalent bonding on permalloy nanoaprticles followed by avidin–biotin interaction to immobilize human serum IgG, as a model protein. Fourier transform infra-red spectroscopy (FTIR) and lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) have been used to investigate the immobilization of human serum IgG on Ni80Fe20 nanoparticles.
Preparation of Ni80Fe20 permalloy nanoparticles
N80Fe20 permalloy nanoparticles with average particle diameter of 71 nm (hereafter denoted as N80Fe20 PNPs) were synthesized by polyol processing described elsewhere (Qin et al. 2009). Briefly, 0.1 M FeCl2, 0.1 M NiCl2, and 2 M NaOH were dissolved in 100 mL propylene glycol at 80 °C by mechanical stirring, and then heated to 180 °C for 2 h. The particle sizes were controlled with the proper addition of K2PtCl4 solution. The resulting product was centrifuged and washed with acetone, alcohol, and distilled water for several times and then dispersed in alcohol. The scanning electron microscopy image of the N80Fe20 PNPs and their magnetic loop at room temperature are shown in Fig. 1S (Please see the Supporting Information).
Biotinylation of human serum IgG
180 μL of deionized water was added into 1 mg sulfo-NHS-LC Biotin (Pierce®) and mixed well to give a final concentration of 10 mM. 28 μL of freshly prepared 10 mM biotin was added to 500 μL of 4.2 μg/μL IgG (from human serum, Sigma). The mixture was incubated on ice for 2 h. After incubation, biotinylated IgG was purified using Zeba Spin column (MWCO 50 kDa, Thermo Scientific, USA). The degree of biotinylation was then determined using Pierce Biotin Quantification Kit (Thermo Scientific, USA).
Immobilization of human serum IgG onto N80Fe20 PNPs
Sketch of modification of N80Fe20 PNPs
The attenuated total reflection FTIR spectra were obtained using a Thermo Nicolet 5700 spectrometer. The measurement was based on 32 scans with a resolution of 4 cm−1. For gel electrophoresis, samples were loaded on NuPAGE Novex 4-12% Bis–Tris Gels (Invitrogen, Australia). The gel was stained with SYPRO Ruby Protein Stains according to the manufacturer’s instruction for analysis. The gel was read using TyphoonTM 9400 Variable Mode Imager.
Results and discussion
Human serum IgG was attached on the surface on N80Fe20 PNPs using a two-step process as shown in Fig. 1, where His-tagged streptavidin was firstly immobilized via non-covalent ligand binding. The functionalization of N80Fe20 nanoparticles with streptavidin is advantageous because of its extremely high affinity interaction with biotin, whereas biotin can be easily conjugated to different proteins so that a variety of assays can be developed for detection of different analytes (Darain et al. 2003; Cai Qi et al. 2009; LeVine 2006).
An important parameter of streptavidin functionalization is the biotin-binding capacity of the modified permalloy nanoparticles. After immobilization of His-tagged streptavidin on N80Fe20 PNPs, the biotin-binding capacity of immobilized streptavidin was measured using HABA (4′-hydroxyazobenzene-2-carboxylic acid) assay as described by Janolino et al. (1995). In this method, immobilized streptavidin is reacted with excess biotin and the unbound biotin is quantified spectrophotometrically. Absorbance of the HABA–avidin complex at 500 nm was measured before and after addition of the sample supernatant or the standard control. Then, the amount of biotin-binding capacity was obtained from the difference between the decrease in absorbance at 500 nm of the standard control and the sample using the molar absorptivity (Janolino et al. 1995). It was found that the biotin-binding site of immobilized His-tagged streptavidin is 0.0886 ± 0.02 (n = 3) μmol per mL of permalloy nanoparticles suspension (1.772 ± 0.004 (n = 3) nmol per mg of dry N80Fe20 PNPs). A blank experiment was also carried out in which free N80Fe20 PNPs without His-tagged streptavidin, to investigate whether any biotin attached on permalloy nanoparticles non-specifically. No absorbance change was observed between sample supernatant and standard control at 500 nm indicating that there was no non-specific adsorption of biotin.
The amount of immobilized His-tagged streptavidin was calculated using HABA method assuming that all of the immobilized streptavidins have biotin-binding sites, by multiplying the concentration of obtained biotin-binding sites with the molecular weight of His-tagged streptavidin. The result showed that 0.0302 ± 0.006 (n = 3) mg of His-tagged streptavidin was immobilized per milligram of dry Ni80Fe20 nanoparticles, which is in good agreement with that (0.034 ± 0.004 (n = 3) mg His-tagged streptavidin per milligram of dry PNPs) obtained by measuring absorbance at 280 nm using NanoDrop spectrophotometer.
To demonstrate the ability of the newly functionalized permalloy nanoparticles to bind biologically relevant substrates, we chose human serum immunoglobulin (IgG) as a model substrate. In order to immobilize human IgG on N80Fe20 PNPs through high affinity avidin–biotin interaction, biotinylation of IgG was carried out. Human serum IgG was conjugated with Sulfo-NHS-biotin via the amine group of IgG. The mole-to-mole ratio of biotin to IgG was investigated using Pierce® Biotin Quantification Kit. Based on the measurements of UV absorbance at 500 nm, 4.5 biotin molecules in average were successfully attached on each IgG molecule.
Another control experiment (control 3) was carried out to further investigate whether biotinylated human IgG binding to Ni80Fe20 permalloy has any effect of non-specific protein binding. For this study, free Ni80Fe20 PNPs (omitting the incubation step with His-tagged streptavidin) were treated with biotinylated human IgG under identical condition. It is worthy to notice that no band was appeared for IgG as shown in Lane 8 in Fig. 3, indicating that there was no non-specific protein adsorption on to Ni80Fe20 nanoparticles. Thus, the above results confirm that human serum IgG was successfully anchored on Ni80Fe20 PNPs via high affinity interactions.
An efficient and convenient method of functionlization of a novel Ni80Fe20 permalloy nanoparticles with human serum IgG was demonstrated by means of a two-step process for the first time. In this process, histidine-tagged streptavidin was first immobilized on to the Ni80Fe20 particles via non-covalent binding followed by IgG binding achieved through high affinity interaction of avidin and biotin. We do believe that this approach may open a new avenue for anchoring proteins and antibodies to Ni80Fe20 nanoparticles via an oriented assembly of proteins on to it.
The described bio-functionalized Ni80Fe20 permalloy nanoparticles system can find potential applications in clinical diagnostics for highly sensitive and multiplexed detection of protein or nucleic acid biomarkers associated with specific diseases. For example, the bio-barcode assay invented and reported by Mirkin et al. (Stoeva et al. 2006) for the multiplexed detection of cancer biomarkers can benefit from the permalloy nanoparticles. The assay involves two types of particles (Stoeva et al. 2006). The first is magnetic microparticle conjugated with antibody to capture the target. The second is gold nanoparticle conjugated with recognition antibody and thiolated single-stranded barcode DNAs. The sandwich complexes are isolated with magnetic field and barcode strands are released and identified. In its published format, the current bio-barcode assay utilizes micrometer-sized magnetic particles; the nanometer-sized Ni80Fe20 permalloy particles are advantageous as they have high surface area to volume compared to micrometer-sized particles, while at the same time retaining high magnetic moment per particle, due to the higher magnetic permeability of permalloy. The Ni80Fe20 nanoparticles are well dispersive in aqueous solution with minimal aggregation and have very high magnetic susceptibility to trigger robust responses to external magnetic field. In view of these merits, it is believed that the novel bio-functionalized nanometer-sized Ni80Fe20 permalloy particles will be potentially applicable in automated multiplexed biomarker detection assays. Current work is underway to test and implement of this novel permalloy biomedical nanomaterials toward clinical diagnostics.
This work was financially supported by the Innovation Projects Fund National and International Research Alliances Program of Queensland Government, Australia. This work was in part supported by Key Project of Science and Technology, the Ministry of Education of China (No. 108039).