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.
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