Biomolecule Immobilization onto Plasma-Functionalized GraphiteEncapsulated Magnetic Nanoparticles for Medical Application Masaaki Nagatsu, Teguh Endah Saraswati, Kosuke Kawamura, Akihisa Ogino Graduate School of Science and Technology, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu, 432-8561, Japan
[email protected] Abstract: The graphene layer-encapsulated iron nanoparticles were modified by pre-treatment of Ar plasma and post-treatment of NH3 plasma using an inductively coupled RF plasma for medical application, such as drug delivery system. Analysis of XPS spectra have been carried out to study the effect of the plasma treatment on the improvement of enrichment of nitrogen-containing groups together with Energy Dispersive X-Ray Spectroscopy elemental mapping to observe the distribution of elements. After chemical modification of amino group onto the surface of nanoparticles by NH3 plasma, we carried out the derivertization method and fluorescent labeling technique to analyze amino group introduction and demonstrated the immobilization of dextran onto the aminated surface of nanoparticles. Keywords: nanoparticles, RF plasma, biomolecule immobilization, surface modification, medical application
1. Introduction Magnetic nanoparticles (MNPs) have many great interests in potential to bio-application such as drug delivery system, hyperthermia treatments, magnetic resonance imaging contrast enhancement, etc.[1-7] So far, these particles have been produced by conventional arc-discharge, modified arc-discharge, chemical vapor deposition (CVD), etc. Among them, the graphene layer-encapsulated MNPs (GEMNPs), fabricated by dc arc-discharge, can leave the toxicity out without detracting their magnetic properties and ensure the biocompatibility required in the medical applications. As for the bioapplication, the introduction of amino groups composed of primary amines to the particles surface achieves enhanced wettability and improves its adhesion. Recently, there are several papers studying about the amino functionalization for carbon nanotubes[8, 9], amorphous carbon sheet[10], nanocrystalline diamond[11], carbon nanoparticles[12], etc. However, this modification has not been deeply studied on carbon encapsulated magnetic nanoparticles. In fact very few information can be
found on the topic of graphene layer-encapsulated iron nanoparticles related to the plasma surface treatment in order to introduce nitrogen-containing group functionalities, such as amino group. In this study, we mainly functionalize the GEMNPs using Ar and NH3 plasma performed by inductivelycoupled RF plasma. After plasma treatment, the biomolecules are immobilized to the particles to test the role of the nitrogen-containing group as a linker to the biomolecules. So far, it has been reported that a variety of bioactive molecules, such as DNA, protein A, hyaluronic acid, heparin, immunoglobulin G, enzymes such as glucose oxidase and glucose isomerase, lysozyme, and polysaccharides such as dextran and carboxylmethyl-dextrans have been successfully immobilized onto the amino-group introduced surface of nanoparticles[13]. Here, we employed dextran as the biomolecules to be immobilized onto the aminated surface of GEMNPs. To study the amino group addition quantitatively and qualitatively, the derivertization method and fluorescent labeling technique were used. The x-ray diffraction(XRD), XPS, high resolution TEM(HR-
TEM) and Energy Dispersive X-Ray Spectroscopy (EDS) elemental mapping were used to characterize the various properties of modified GEMNPs.
2. Experimental The graphene layer-encapsulated iron particles were prepared by using arc discharge method referred to Ref. [14]. The arc discharge was generated by applying a dc current of 150-200 A at about 20 V between anode and cathode. Graphite electrodes molded with Fe2O3 powder was used as anode. In the other side, graphite rod was used as cathode. Both of those electrodes were set with distance as near as possible in a stainless-steel vacuum chamber with 200 mm diameter. The chamber was evacuated to around 1 Pa by a rotary pump. A mixture gas of He:CH4 with ratio 8:2 was flown to the chamber until the pressure reached 1.3 x10 4 Pa. Then, carrying a high current between the electrodes will provide lots of composite powders. The powders were directly deposited on silicon substrates set inside the chamber. For structural characterization, observation using HR-TEM was performed by using a JEM-2100F (JEOL) equipped CCD camera. The TEM sample was prepared by dispersing the
Fe
Graphene Fig. 1 HR-TEM images of magnetic iron nanoparticles coated by graphene layers[14]
desired particles in ethanol and dropped onto the carbon grid. The TEM observation was conducted at an accelerating voltage of 200 kV. As shown in Fig. 1, the diameters of particles are around 10-50 nm in size measured from the outmost graphene layer[15]. The spacing of the graphene layers is about 0.34 nm. High resolution of α-Fe interplanar distance, 0.207 nm, is clearly observed in core particle region shown in Fig.1. The nanoparticles are treated by using an inductively-coupled radio frequency plasma device. The schematic view of the chamber is shown in Fig. 2. The water-cooling copper pipe helical antenna, 100 mm in coil diameter and 20 mm in pipe diameter, was coupled to an RF power generator at 13.56 MHz via a matching net-work. Typical input RF power was about 80 W. Samples were set in the glass dish placed on the stage inside the chamber. The pretreatment was performed with Ar plasma and subsequently NH3 gas plasma was used as the posttreatment to introduce the amino groups.
3. Results and discussion In order to investigate the effect of chemical modification on the plasma-treated surfaces by Ar and NH3 plasma, XPS measurements were carried out. The relative compositions of C 1s, N 1s, O 1s and Fe 2p, and atomic ratios of O/C and N/C of the samples before and after plasma treatment under different plasma conditions are listed in Table 1. The experimental results show that the relative composition of C 1s decreased after plasma treatment due to the ion bombardment of Ar plasma pretreatment. With the Ar plasma pre-treatment, many free carbon bonding are expected to be created in the outmost of graphene layer and then react directly Table 1. Atomic composition of C 1s, O 1s, N 1s and Fe 2p peaks and atomic rations of O/C and N/C taken from the XPS spectra before and after plasma treatment under various plasma conditions
Fig.2
Inductively coupled RF plasma device.
with NH3 plasma to produce an aminated surface during the post-treatment. Based on the XPS results, there is a significant peak can be observed at around 400.0 eV in the N 1s spectra after ammonia plasma treatment, which is possibly identified as nitrogencontaining functional group, such as -C-NH2. The highest atomic percentage of nitrogen is 4.40% increased from 0% of the control sample (untreated). Figure 3a represents to a STEM image of the treated sample while the four images (Figs. 3b-3e) represent to the EDS elemental mapping of C, Fe, O and N elements, respectively. The contrast color represents to the each element. The results of N mapping show that a contrast area exists over the whole area of particles but in less contrast. It indicates that the nitrogen element is found on the surface area of the particles, which means that the surface modification successfully attach the nitrogen-containing groups on the outmost of particle surfaces. From the dominant signal at around 399.9 eV in N 1s region found in XPS spectra, it is considered that they are attributable to amino group.
(a)
(b)
(c)
group derivatization. Dextrans serve as one of the most promising macromolecular carrier candidates for a wide variety of therapeutic agents due to their excellent physico-chemical properties and physiological acceptance. Being very hydrophilic, dextran will provide highly hydrated coatings in contact with an aqueous medium, and their wide range of compositions and structures can be used to pursue various aims. Moreover, dextran can be activated at multiple sites throughout its chain, since each monomer contains hydroxyl resides. Therefore, polyaldehyde dextran can be used to couple many small molecules, such as drugs, to a targeting molecule like an antibody. The schematic step of immobilization is illustrated in Fig. 4.
RF Plasma treatment (Ar, NH3)
Fe (d)
= Amino-dextran backbone)
H2N NH2
H2N
+
NH2 NH2
oxidized dextran
NH2
+H2O -H2O H2N H2N
(e)
NH2 NH2
Recent experimental results of fluorochromelabelling technique using fluorescent dye (SDP ester) showed that the surface of GEMNPs were uniformly aminated by a series of Ar plasma pretreatment followed by NH3 plasma post-treatment. Based on the above results, the grafted-amino groups on the GEMPNs surface are expected to be a potential covalent bonding to biomolecules. Here, we investigated the immobilization of dextran coupled by drug molecules, following with amino
NH2
NaBH4
N
Fig. 3 STEM image (a) and EDS elemental mapping images (b, c, d, e) of C, Fe, O and N elements, respectively, of treated sample with condition: 10 min of Ar plasma pre-treatment continued with 2 min of NH3 plasma post-treatment performed at 80 Watt of RF power and 50 Pa of gas pressure.
NH2
H2N H2N
O
(
/ drug molecules
H2N
H2N
C
Dextran
GEMNPs
H2N
H2N
H2N H2N NH2
NH2
Immobilized GEMNPs
Fig. 4 The schematic illustration of dextran molecules immobilization onto the graphite-encapsulated magnetic nanoparticles.
In amino group derivatization method using 3trifluoromethyl benzaldehyde (TFBA), the free amino groups after immobilization can be evaluated quantitatively by analyzing XPS spectra on F 1s peak region (~689 eV binding energy). Figure 5 shows the preliminary experimental results of XPS
analyses for different dextran weights. It is found that by increasing the dextran weight, the free amino groups are decreasing and bound amino groups to dextran are increasing. When dextran weight was 0.2 g, about 80 % of the amino groups introduced on the GEMNP surfaces were used to link dextran molecules. #
##
H2N
H2N
Amino groups (%)
H2N
NH2
*
#
H2 N
#
*
NH2
NH2
#
NH2
#
Bound amino groups ( )
Free amino groups ( # )
*
Dextran weight (gr) Fig. 5 Percentage of free and bound amino group on the GEMNP surfaces for different dextran weights.
Based on these results, the grafted-amino groups on the GEMPNs surface are expected to be a potential covalent bonding to biomolecules. In the conference, we will present the results on the effect of substrate biasing during RF plasma treatment of GEMNPs on the efficiency of amino group introduction.
4. Conclusion Based on the results and discussion described above, it can be concluded that the iron encapsulated inside graphite layer is successfully synthesized by arc discharge method. After the plasma treatment, the surface of the outmost graphene layer is successfully covered by nitrogen-containing groups due to the XPS spectra and the STEM-EDS elemental mapping show the definitive assignment of nitrogen element group attached on the outmost of the particle surface. In the further step, the grafted amino groups on the treated GEMNPs are successfully used as linker to immobilize the dextran.
Acknowledgement This work has been supported in part by the Grantsin-Aid for Scientific Research and performed under Research and Education Funding for Research Promotion supported by MEXT.
References [1] Q. A. Pankhurst, N. K. T. Thanh, S. K. Jones, J. Dobson: J. Phys. D: Appl. Phys., 42 (2009), p.224001. [2] C. C. Berry: J. Phys. D: Appl. Phys., 42 (2009), p.224003. [3] Q. A. Pankhurst, J. Connolly, S. K. Jones, J. Dobson: J. Phys. D: Appl. Phys., 36 (2003), p.R167. [4] G. Pastorin: Pharmaceutical Research, 26 (2009), p.746. [5] S. Kim, E. Shibata, R. Sergiienko, T. Nakamura: Carbon, 46 (2008), p.1523. [6] A. Ito, M. Shinkai, H. Honda, T. Kobayashi: J. Biosci. Bioeng., 100 (2005), p.1. [7] C. B. Catherine, S. G. C. Adam: J. Phys. D: Appl. Phys., 36 (2003), p.R198. [8] C. Chen, B. Liang, D. Lu, A. Ogino, X. Wang, M. Nagatsu: Carbon, 48 (2010), p.939. [9] A. Felten, C. Bittencourt, J. J. Pireaux, G. Van Lier, J. C. Charlier: J. Appl. Phys., 98 (2005), p.074308. [10] N. Inagaki, K. Narushima, H. Hashimoto, K. Tamura: Carbon, 45 (2007), p.797. [11] Z. Remes, A. Choukourov, J. Stuchlik, J. Potmesil, M. Vanecek: Diam. Relat. Mater., 15 (2006) , p.745. [12] W.-K. Oh, H. Yoon, J. Jang: Diam. Relat. Mater., 18 (2009), p.1316. [13] K.S. Siow, L. Britcher, S. Kumar, H.J. Griesser, Plasma Processes Polym. 3 (2006) 392. [14] M. Nagatsu, T. Yoshida, M. Mesko, A. Ogino, T. Matsuda, T. Tanaka, H. Tatsuoka, K. Murakami: Carbon, 44 (2006), p.3336. [15] T. E. Saraswati, T. Matsuda, A. Ogino, M. Nagatsu: Diam. Relat. Mater., 20 (2011) p. 359.
