In Situ hybridization of superparamagnetic iron-biomolecule nanoparticles

Article abstract of DOI:10.1021/ja505242c

The increase in interest in the integration of organic-inorganic nanostructures in recent years has promoted the use of hybrid nanoparticles (HNPs) in medicine, energy conversion, and other applications. Conventional hybridization methods are, however, often long, complicated, and multistepped, and they involve biomolecules and discrete nanostructures as separate entities, all of which hinder the practical use of the resulting HNPs. Here, we present a novel, in situ approach to synthesizing size-specific HNPs using Fe-biomolecule complexes as the building blocks. We choose an anticancer peptide (p53p, MW 1.8 kDa) and an enzyme (GOx, MW 160 kDa) as model molecules to demonstrate the versatility of the method toward different types of molecules over a large size range. We show that electrostatic interaction for complex formation of metal hydroxide ion with the partially charged side of biomolecule in the solution is the key to hybridization of metal-biomolecule materials. Electrochemical deposition is then used to produce hybrid NPs from these complexes. These HNPs with controllable sizes ranging from 30 nm to 3.5 ?m are found to exhibit superparamagnetic behavior, which is a big challenge for particles in this size regime. As an example of greatly improved properties and functionality of the new hybrid material, in vitro toxicity assessment of Fe-GOx HNPs shows no adverse effect, and the Fe-p53p HNPs are found to selectively bind to cancer cells. The superparamagnetic nature of these HNPs (superparamagnetic even above the size regime of 15-20 nm!), their biocompatibility, and the direct integration approach are fundamentally important to biomineralization and general synthesis strategy for bioinspired functional materials.

Full text of DOI:10.1021/ja505242c

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Article
In-situ Hybridization of Superparamagnetic Iron-Biomolecule Nanoparticles
Nafiseh Moghimi, Apraku David Donkor, Mamata Mahapatra, Joseph
Palathinkal Thomas, Zhengding Su, Xiaowu Shirley Tang, and K.T. Leung
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja505242c • Publication Date (Web): 03 Jul 2014
Downloaded from http://pubs.acs.org on July 8, 2014
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In-situ Hybridization of Superparamagnetic Iron-
Biomolecule Nanoparticles
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Nafiseh Moghimi,1 Apraku David Donkor,1 Mamata Mahapatra,1,2 Joseph Palathinkal
Thomas,1 Zhengding Su,3 Xiaowu (Shirley) Tang,1 K.T. Leung1,*
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1
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L3G1
Department of Hydro & Electro Metallurgy, Institute of Minerals and Materials
Technology, Council of Scientific & Industrial Research, Bhubaneswar - 751 013, Odisha,
India
Danny Thomas Research Center, St Jude Children's Research Hospital, Memphis, TN, USA
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38103
* Corresponding author. Eꢀmail: tong@uwaterloo.ca
ABSTRACT
The increase in interest in the integration of organicꢀinorganic nanostructures in recent years
has promoted the use of hybrid nanoparticles (HNPs) in medicine, energy conversion and
other applications. Conventional hybridization methods are, however, often long,
complicated and multiꢀstepped, and they involve biomolecules and discrete nanostructures as
separate entities, all of which hinder the practical use of the resulting HNPs. Here, we
present a novel, inꢀsitu approach to synthesizing sizeꢀspecific HNPs using Feꢀbiomolecule
complexes as the building blocks. We choose an anticancer peptide (p53p, MW 1.8 kDa) and
an enzyme (GOx, MW 160 kDa) as model molecules to demonstrate the versatility of the
method towards different types of molecules over a large size range. We show that
electrostatic interaction for complex formation of metal hydroxide ion with the partially
charged side of biomolecule in the solution is the key to hybridization of metalꢀbiomolecule
materials. Electrochemical deposition is then used to produce hybrid NPs from these
complexes. These HNPs with controllable sizes ranging from 30 nm to 3.5 ꢁm are found to
exhibit superparamagnetic behaviour, which is a big challenge for particles in this size
regime. As an example of greatly improved properties and functionality of the new hybrid
material, in vitro toxicity assessment of FeꢀGOx HNPs shows no adverse effect and the Feꢀ
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p53p HNPs are found to selectively bind to cancer cells. The superparamagnetic nature of
these HNPs (superparamagnetic even above the size regime of 15ꢀ20 nm!), their
biocompatibility, and the direct integration approach are fundamentally important to
biomineralization and general synthesis strategy for bioinspired functional materials.
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Keywords: Biocompatibility, targeted delivery, FeꢀGOx, anticancer p53 peptide, inꢀsitu
hybridization, superparamagnetic nanoparticles
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Introduction
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Composite systems of biomolecules and metal nanostructures combine the evolutionꢀ
optimized molecular recognition capability of biomolecules with the novel optical, electronic,
and catalytic properties of nanomaterials. These composite systems offer excellent prospect
for developing a new generation of multiꢀfunctional bioelectronic devices. In the past
decade, there has been substantial progress in the use of biomoleculeꢀnanostructure
composites in electrochemical biosensors, electronic nanocircuitry, and even nanodevices.1–4
In conventional approaches, the biomolecules and metal nanostructures are treated as separate
entities. Bioꢀintegration into nanostructures could only be obtained by immobilization of
biomolecules on the inner and/or outer surfaces of the nanostructures, including polymer
matrices, inorganic supports and nanoparticles (NPs).5–7 Wellꢀestablished techniques based
on physical adsorption, electrostatic binding, specific recognition, and covalent coupling have
often been used for immobilization.8–11 In particular, adsorption of proteins on NPs has been
achieved through electrostatic interactions between the partially charged amino acid side
groups of the protein and the appropriate component with opposite polarity on the NPs. The
latter component could be a functional group of a linker molecule preꢀcoated on the NPs or a
partially charged site on the nanostructure.12,13 There is a general lack of study on bioꢀ
integration into the nanostructure during growth. Such studies can provide fundamental
information about the nature of this integration (including bonding, interactions, and growth
mechanism), which could lead to a new class of bioꢀnanomaterials. With many applications
in biosensor research, food industry, and renewable fuel cells,14–18 glucose oxidase (GOx,
with MW of 160 kDa) is a popular model biomolecule for studying selfꢀorganization of
nanostructures on biomolecules and of biomolecules on other surfaces. In the limited number
of studies on conjugating GOx onto NPs by electrostatic interaction,19,20 the NPs were
synthesized first and GOx were subsequently allowed to interact with the NPs. Even in the
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earlier work on the coꢀelectrodeposition of GOx and gold NPs,21,22 separate GOx and preꢀ
grown NPs were used. To date, direct incorporation of biomolecules into the nanostructures
during growth remains challenging, because the presence of an aqueous medium with a mild
pH (as required by the biomolecules) and the need for low growth temperature (preferably
below room temperature) are not conducive to nanostructure growth. Although the chelation
and complexation of metal ions with GOx have been widely studied, there is no report on
using metal ionsꢀGOx complexes directly to synthesize hybrid nanoparticles (HNPs).
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The same hybridization approach can also be easily extended to incorporate other
biomolecules, even with dramatically different sizes, in these HNPs. The anticancer p53
peptide (p53p, with MW of 1.8 kDa) is a promising drug for most cancers because it can bind
to both Mdm2 and MdmX in cancer cells.23 Almost 50% of cancers are caused by
overexpressed Mdm2 and MdmX as they inhibit the tumor suppressor, p53p.24,25 Attaching
p53p to magnetic NPs that can be delivered to targeted tumor sites is a breakthrough in
anticancer therapeutics.
In this work, we report the successful synthesis of FeꢀGOx and Feꢀp53p HNPs by
using FeꢀGOx and Feꢀp53p complexes directly as the building blocks, without involving preꢀ
grown Fe NPs followed by biomolecule coating. We further demonstrate that these HNPs
consist of Fe nanocrystallites binding together by biomolecules similar to a stoneꢀandꢀmortar
architecture. We make these Feꢀbiomolecule complexes by adjusting the pH of the aqueous
solution to above the isoelectric point of GOx or p53p, in which the negatively charged
amino acids on the surface of the biomolecule electrostatically interact with the positive Fe
ions. By conducting the coꢀelectrodeposition at 4°C, we produce FeꢀGOx and Feꢀp53p HNPs
that show superparamagnetic properties even when these HNPs are as large as a few hundred
nanometers, while preserving the bioactivities of GOx and p53p at the same time. The Feꢀ
GOx HNPs also exhibit remarkable biocompatibility to human hepatoma cells (liver cells).
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This method of producing sizeꢀspecific superparamagnetic HNPs has the important advantage
of attaining the desired size while eliminating the postꢀfunctionalization (coating) step to
achieve the size needed for practical use of these superparamagnetic NPs. We show that in
the present hybridization method GOx not only maintains its enzymatic activities but also
acts as a protective shell from oxidation for metallic Fe nanocrystallites. As a further
practical demonstration, we also show that p53p remains active after hybridization and can
bind to Mdm2, which confirms the generality of the present approach. Isolation of Fe
nanocrystallites by GOx or p53p introduces extraordinary magnetic property for large hybrid
nanoparticles (i.e. superparamagnetism in large sizes). Electrochemical hybridization of
metals with biomolecules therefore offers a novel approach to developing cellꢀfriendly
magnetic HNPs with homogeneously distributed magnetic nanocrystallites and biomolecules
inside. The present method is however not limited to electrochemical reduction, and Feꢀ
biomolecule complex reduction with any other reducing agent would also be possible. These
HNPs promise potential applications not only as magnetic contrast agents but also in targeted
slowꢀrelease drug delivery systems.
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Materials and Methods
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Preparation of the HNPs: FeꢀGOx HNPs were prepared by coꢀelectrodeposition of
Fe and GOx on a Hꢀterminated Si(100) substrate in a threeꢀelectrode cell using an
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electrochemical workstation (CH Instruments 660A). A silicon chip (15×2.5 mm2, 0.4 mm
thick), precut from a Si(100) wafer (pꢀtype, Bꢀdoped, with a resistivity of 0.01ꢀ0.02 ꢂꢀcm),
was used as the working electrode after it was cleaned using the RCA method. It was then Hꢀ
terminated by dipping in a 2% aqueous HF solution. A standard Ag/AgCl electrode was used
as the reference electrode and a Pt wire as the counter electrode. The electrolyte consisted of
5 mM FeCl2 by dissolving FeCl2⋅6H2O salt (Aldrich, 99%) in an aqueous solution with 200
mM boric acid as the supporting electrolyte. The pH of the electrolyte solution was then
adjusted to 6.5 with 250 mM NaOH (Aldrich, 99.9%). After deoxygenation with N2 for 30
minutes, the electrolyte was mixed with GOx from Aspergillus niger (Fulka) to obtain 75 ꢁM
GOx in the solution. In this freshly prepared electrolyte solution, electrodeposition was
conducted at 4 °C by amperometry potentiostatically at −1.2 V vs Ag/AgCl for an appropriate
deposition time. The resulting electrodeposits were washed thoroughly with filtered
deionized water (18.2 Mꢂ•cm) and stored in N2 at room temperature for further analysis. Feꢀ
p53p HNPs were prepared using the same method described above for FeꢀGOx HNPs. The
p53p peptide, with the sequence of SQETFSDLWKLLPEN,26 was chemically synthesized
using standard Fmoc solidꢀphase chemistry27 at St Jude Children’s Research Hospital Peptide
Facility. The synthetic peptides were purified by HPLC using a reverseꢀphase C18 semiꢀ
preparative column with an acetonitrile gradient of 10–35%. The purity of the peptides was
verified by a reversedꢀphase analytical HPLC column and the identity of the final products
was verified by mass spectral analysis. The isoelectric point of p53p is 4.0, which is similar
to that of GOx (4.2). The same concentration of p53p (as that of GOx i.e. 75 ꢁM) was added
to a solution of 5 mM FeCl2 with a pH of 6.5. The electrodeposition was also carried out at 4
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°C followed by a thorough wash with filtered deionized water and kept at 4 °C for bioactivity
tests.
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Analysis of the HNPs: The surface morphology of the soꢀprepared samples of Feꢀ
GOx and Feꢀp53p HNPs were characterized by fieldꢀemission scanning electron microscopy
(SEM) (Zeiss LEO FEꢀSEM 1530 microscope) and by Helium ion microscopy (HIM) (Zeiss
Orion Plus microscope). Multiꢀcrossꢀsectional imaging was conducted using a Zeiss Auriga
focused ion beam (FIB) SEM system. The samples were first coated by a thin carbon layer to
protect the top surface from damage due to the sputtering Ga ion beam. Very thin slices of
the samples with thickness no larger than 1 ꢁm (containing selected NPs on top of the Si
substrate) were cut vertically along the surface normal (i.e., in the depth direction of the
substrate). SEM images of the exposed cross sections were then collected by using the
backscattered electron detector in order to differentiate elements with different atomic masses
based on the image contrast. Subsequent crossꢀsectional SEM images were collected
interleavingly after removing additonal vertical slices (10 nm thick) from the samples.
Crystal structures of HNPs were also determined by glancingꢀincidence Xꢀray diffraction
(XRD) (PANalytical X’Pert Pro MRD diffractometer) with Cu Kα (1.542 Å) radiation at an
incidence angle of 0.5°.
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Chemicalꢀstate composition of the HNPs were analyzed as a function of Ar ion
sputtering time by depthꢀprofiling Xꢀray photoelectron spectroscopy (XPS) (ThermoꢀVG
Scientific ESCALab 250 microprobe), equipped with a monochromatic Al Kα radiation
(1486.6 eV) and operated with a typical energy resolution of 0.4ꢀ0.5 full width at halfꢀ
maximum (fwhm). Threeꢀdimensional chemical mapping of the HNPs were conducted by
timeꢀofꢀflight (TOF) secondary ion mass spectrometry (SIMS) (IONꢀTOF SIMSꢀ5 system),
equipped with a 2 m long TOF reflectron analyser and a multichannel detector along with a
+
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Bi3 liquid metal analysis ion source and an O2 sputtering ion source. More details on the
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chemical mapping procedure can be found in the Supporting Information. For the kinetics
studies, absorption spectra were collected using a PerkinꢀElmer Lambda 35 UVꢀVisible
spectrometer equipped with a Labsphere integrating sphere. Magnetic properties were
studied in a Quantum Design Dynacool vibrating sample magnetometer (VSM).
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Cellular Assay: To test the biocompatibility of the HNPs, cells were cultured on
substrates with attached HNPs. Before the cellular assays, the HNPs were sterilized with 70%
ethanol. HeLa cells (HeLa, No: CCLꢀ2; ATCC)) were cultured in Dulbecco’s modified
Eagle’s medium with 10% fetal bovine serum and 1% penicillinꢀstreptomycin (ATCC). Cells
were maintained in 5% CO2 in humidity at 37°C. For live cell imaging experiments, 1ꢀ2×104
cells were seeded on glass cover slips and allowed to attach overnight. The cells were then
cultured and observed for several days.
Selective binding of Mdm2-GST to Fe-p53p:
A Mdm2ꢀGST solution with concentration of 61 ꢁM was prepared to check the
binding of p53pꢀMdm2 after hybridization. Details about the preparation of Mdm2 and
Mdm2ꢀGST are given in the Supporting Information. The binding of p53p with Mdm2 is
sufficiently strong that no separation was found even upon vigorous washing during
synthesis. The presence of Mdm2 could therefore be revealed in the corresponding SEM
images.
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Results and Discussion
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FeꢀGOx HNPs was prepared at 4 °C by coꢀelectrodeposition of Fe and GOx on a Hꢀ
terminated Si(100) substrate in a threeꢀelectrode cell. The electrolyte solution consisted of 5
mM FeCl2 mixed with an appropriate amount of GOx from asparagines to obtain a
concentration of 75 ꢁM GOx in a 200 mM boric acid solution. In order to take advantage of
the electrostatic attraction between GOx (with an isoelectric point of 4.2) and Fe2+ ions, the
solution was maintained at pH 6.5 such that GOx became partially negative. UVꢀVisible
spectroscopy was used to monitor the change in the absorption band of GOx (Figure S1a,
Supporting Information). The Fe electrolyte (i.e., Fe2+) exhibits a featureless spectrum with
no discernible peaks in the region of interest, while the GOx spectrum shows a strong, sharp
absorption peak at 274 nm along with weaker, broader bands at 377 nm and 454 nm, all of
which correspond to the oxidized form of flavin groups.28,29 The FeꢀGOx electrolyte,
however, exhibits a peak at 273 nm with a shift in the broad bands to 383 nm and 452 nm,
respectively.
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Formation of GOxꢀFe complex is possible because of the many functional groups,
such as amine, imidazole and sulfhydryl groups, in GOx that could interact with oxidizing
mediators. Although both cationic and anionic groups are located on the GOx surface, the
enzyme molecule has an essentially negative electrostatic surface potential at pH 6.5 as a
result of excess glutamate and aspartate over lysine and arginine.30,31 The presence of this
negative surface potential favours electrostatic complexation of the anionic sites of the
enzyme with mediators containing positively charged groups. In an aqueous environment,
FeCl2 tends to form FeOH+ monomers,32–34 with a significantly higher ionic mobility than the
considerably larger GOx molecule. The resulting FeOH+ could therefore attach to the anionic
sites of GOx through a chargeꢀtransfer bond.
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To further investigate the FeꢀGOx complexation, we performed cyclic voltammetry
on the FeꢀGOx electrolyte (Figure S1b, Supporting Information), and compare the result with
those on the pristine GOx electrolyte and Fe electrolyte in UV experiments (Figure S1a,
Supporting Information). In the absence of GOx, oxidation of Fe2+ to Fe3+ complex species
occurs at an anodic peak potential of 2 mV (vs Ag/AgCl), while reduction occurs at a
cathodic potential of –450 mV (vs Ag/AgCl) upon the reverse scan. In the presence of GOx,
both anodic and cathodic peak potentials are shifted to more positive values, with the shift in
the cathodic peak potential (from –450 mV to –370 mV) found to be discernibly larger than
that of the anodic peak potential (from 2 mV to 5 mV). The smaller reduction potential
needed for Fe3+ to Fe2+ in the presence of GOx confirms the complex formation of the Fe
species that changes the reduction potential, in good accord with our UVꢀVisible
spectroscopic results. A decrease in the corresponding current density is also observed,
which can be attributed to the slower diffusion of FeOH+ when bound to a large and slowly
moving GOx molecule. In general, the concentrations of the redox species and the number of
electrons involved in the reactions control their diffusion coefficients and the resulting
current densities.35 As these voltammograms were obtained under the same experimental
conditions, the observed difference in the current density is attributed to the difference in
diffusion coefficients. The diffusion coefficient for FeOH+ is expected to be smaller upon
binding with GOx. A similar observation has been reported for the [FeII(phen)3]2+ when
bounded to GOx and to DNA.36,37
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Using the FeꢀGOx complexes as our precursors, we synthesized the HNPs. In a
freshly prepared FeꢀGOx electrolyte solution, homogenous spherical NPs were deposited on
the Hꢀterminated Si substrate by amperometry at −1.2 V (vs Ag/AgCl). These NPs exhibit a
remarkably narrow size distribution, with the average size easily controllable from 30 nm to
3.5 ꢁm by varying the respective deposition time from 10 s to 60 minutes (Supporting
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Information, Figure S2). Figure 1a shows a typical HIM image of FeꢀGOx HNPs obtained by
a 30 minute deposition. Furthermore, these nanograins are made up of nanocrystallites of 18
nm dia. in size (Figure 1b), as estimated by applying the Scherrer analysis to the Fe(110)
diffraction peak in the corresponding Xꢀray diffraction pattern (Figure 1d). The features in
the XRD pattern (Figure 1d) correspond to the bcc phase of metallic Fe (PDF2 006ꢀ0696).
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To confirm the activity of GOx after electrodeposition, we used a glucose
spectrometric assay to monitor the production of H2O2 by detecting quinoneimine dye (with
details given in Supporting Information, Figure S3). The characteristic absorption band of
the dye at 510 nm is found to become more intense over 15 min, which indicates that GOx
remains bioactive upon hybridization into the NPs. After the bioactivity test, the sample was
washed thoroughly with filtered deionized water, which also makes the porous structure of
the HNPs more obvious (Figure 1c). The appearance of the porous structure suggests
diffusion of glucose into the FeꢀGOx HNPs to reach the active sites of GOx, which causes the
nanograins to cluster into larger grains resulting in a more porous appearance for the NPs.
The average nanocrystallite size also increases from 18 nm to 24 nm as estimated from their
respective XRD patterns (Figure 1d). This suggests the presence of GOx in the deeper layers
of NPs and not just on the surface. It should be noted that the XRD features obtained after
the activity test also correspond to the bcc phase of metallic Fe. We also soaked the FeꢀGOx
HNPs in H2O2 overnight. No increase in porosity or grain clustering was observed, which
confirms that such an increase in porosity is caused by glucose reaction with HNPs, thereby
validating the unique activity of the HNP. No increase in porosity was observed upon the
glucose activity test on the pristine Fe NP sample (i.e. without GOx).
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Figure 1: HIM images of FeꢀGOx hybrid nanoparticles (a) and (b) before and (c) after the
activity test, and (d) their corresponding glancing incidence XRD patterns before (bottom
curve) and after the activity test (top curve). Scale bars represent 5 ꢁm in (a) and 1 ꢁm in (b)
and (c).
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The presence of nanosized grains throughout a HNP can also be observed in the
tomography images obtained by using the multiꢀcrossꢀsectional imaging technique in a FIBꢀ
SEM.38,39 This technique allows us to observe nanostructures buried within the bulk with a
spatial resolution of 10 nm. The SEM image obtained with the backꢀscattered detector in
Figure 2a shows the top view of two FeꢀGOx HNPs, with the lines marking the vertical cut
locations for the selected crossꢀsectional images, shown in Figure 2bꢀf. The black spots
inside the NPs correspond to voids and/or elements with lower masses, likely reflecting the
locations of GOx, among the brighter Fe nanocrystallites. From the series of crossꢀsectional
images, it is evident that the observed HNPs consist of very small Fe subunits of
nanocrystallites separated by voids and/or organic materials. The fairly uniform distribution
of these GOx locations observed inside the nanoparticles leads us to propose a “stoneꢀandꢀ
mortar” model for the hybrid nanoparticle, shown schematically in Figure 2g. In this model,
individual nanograins (in a FeꢀGOx HNP) consist of Fe nanocrystallites “sticking” together
by GOx molecules. Inside a nanograin, GOx could serve as the substrate, providing
nucleation sites for Fe crystallization, which could proceed by reduction of FeOH+ ions
adsorbed on the GOx substrate through its interaction with the partially negative amino acid
components (e.g. glutamate and asparatate) of the GOx. FeOH+ ions undergo adsorption onto
the GOx followed by reduction to metallic Fe, i.e. FeOH+(ads) + H+ + 2e– → Fe + H2O.
However, to distinguish between an actual void and an organic intermediate layer would
require additional analysis by a complementary method, such as threeꢀdimensional chemical
imaging provided by the SIMS technique.
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Figure 2: Backꢀscattered electron SEM images of (a) the top view of two FeꢀGOx hybrid
nanoparticles (HNPs) before crossꢀsectional imaging, and (bꢀf) crossꢀsectional views of
selected vertical cuts at the locations marked in (a). (g) Schematic “stoneꢀandꢀmortar” model
for FeꢀGOx HNPs electrodeposited on a Si substrate.
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One possible configuration for the hybridization of Fe and GOx is that the NPs consist
of nanocrystallites individually covered by GOx. In this configuration, it should be possible
to observe the GOx inside the NP even when all the GOx molecules on the surface of the NP
are removed. To confirm the presence of GOx along with Fe inside the HNPs, we conduct
3D chemical imaging by TOFꢀSIMS. This method enables us to probe the composition of the
bulk materials at different depth, which makes possible studies not just of 3D composition of
biomaterials but also of diffusion of a component into a semiconductor material.40,41 We
have chosen relatively large HNPs (2.5 ꢁm) for this experiment in order to increase the
accuracy of the characterization, but the results are expected to be similar for all sizes. A
300×300 ꢁm2 area of the sample was preꢀcleaned with O2+ sputtering for 5 s before imaging.
The secondary ions were massꢀseparated in a 2 m long TOF reflectron analyser, and the
appropriate secondary ions Fe+, NaNH2+ for GOx, and Si+ were collected over a rastered
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sampling area of 100×100 ꢁm2 to obtain a 2D map. The sample was then sputtered with O2
to remove the sample layerꢀbyꢀlayer at a wellꢀdefined sputtering rate. The 2D maps were
collected interleavingly between the sputtered layers as a function of sputtering depth to
obtain the depth profiles and to construct the corresponding 3D images of the Fe, GOx and Si
components. Figure 3a shows the 3D images obtained for three component ions and their
overlap (total) for a typical sample of HNPs. The presence of the enzyme component only on
the Fe component (i.e. nanoparticle) and not on the bare surface of the Si substrate can clearly
be seen in the corresponding topmost 2D maps shown in Supporting Information, Figure S4.
We also show as insets in Figure 3a a magnified view of a selected area of the topmost 2D
maps marked by a small square in Figure 3a, top rightmost panel. Figure 3b shows the YZ
cross sections of the respective 3D images in Figure 3a for a selected X position. Evidently,
the concurrence of component intensities at the same location shown in Figure 3b further
confirms the presence of both Fe and GOx throughout the NPs, indicating their hybrid nature.
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The YZ crossꢀsectional maps are plotted with an extended Z scale to better illustrate the
details in the depth direction. The similar decreasing trends found for the Fe and GOx
components with depth are also clearly observed in the corresponding depth profiles shown
in Figure S5.
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Figure 3: (a) TOFꢀSIMS 3D images of Si+ (blue), Fe+ (red), and NaNH2+ (corresponding to
GOx; green), and their overlap (total) for FeꢀGOx hybrid nanoparticles (HNPs) on a 100×100
ꢁm2 area of the sample, with the insets depicting their corresponding topmost 2D maps of a
selected FeꢀGOx HNP in this sampling area (marked by small square in top rightmost panel).
The scale bar for the insets is 2 ꢁm. (b) Selected YZ cross sections of the corresponding 3D
images of FeꢀGOx HNPs (at an X location as marked in the image cube in (a), top rightmost
panel). The horizontal (Y) and vertical (Z) scale bars correspond to 2 ꢁm and 200 nm,
respectively.
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The depthꢀprofiling XPS data shown in Figure 4 provides collaborative support for
our SIMS observation. For the asꢀprepared sample, the C 1s peaks at 284.8 eV, 286.2 eV,
and 288.4 eV can be attributed, respectively, to the C–H, C–O, and C=O components of the
amino acid side chains of the polypeptide backbone of GOx.42,43 Similarly, the N 1s features
at 398.9 eV and 400.2 eV correspond to the NH2 and NHC=O components,44,45 respectively.
Upon sputtering for 110 s, dramatic reductions in the intensities of these C 1s and N 1s
components are observed. Additional C 1s feature found at 283.3 eV and N 1s feature at
397.3 eV can be assigned to C–Fe46 and N–Fe features,47 respectively, indicating the
interaction between GOx and Fe nanocrystallites. After 290 s of sputtering, the Fe 2p3/2 peak
at 710.0 eV and the shoulder emerged at 707.1 eV can be attributed to Fe oxides and metallic
Fe, respectively.47 From our sputtering rate, we estimate that this initial sputtering
corresponds to the removal of a 4ꢀ5 nm thick layer of material, consistent with one monolayer
of physisorbed GOx (uniformly distributed on both the HNPs and Si substrate). Continued
sputtering for over 650 s reveals that the relativeꢀintensity reductions of the C–Fe and N–Fe
features parallel those of the other C and N components of GOx and of the metallic Fe. The
similar trends found in the intensity reductions, along with the presence of N and C even after
extended Ar sputtering (into the deeper layers), provide strong support for the intermixing of
GOx with Fe nanocrystallites inside the HNP.
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Figure 4: Depthꢀprofiling XPS spectra of Fe 2p, N 1s, and C 1s regions for the asꢀprepared
FeꢀGOx hybrid nanoparticles and upon Ar sputtering for 50, 110, 290, 410, 650, 890, 1370,
and 2330 s.
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To further investigate the nature of the Fe–C and Fe–N bonds found between GOx
and Fe before formation of the HNP and the role of these bonds in the evolution of the HNP,
we prepared dropꢀcast thin films of electrolytes containing GOx with FeCl2 (FeꢀGOx
electrolyte) and without FeCl2 (GOx electrolyte) before amperometry. Their C 1s and N 1s
spectra are compared with those of HNPs (after 290 s of sputtering) in Figure 5. (It should be
noted that a brief sputtering was used to remove any surface carbonaceous layer often found
due to sample handling in air.) Evidently, the dropꢀcast film of the FeꢀGOx electrolyte
exhibits all the C 1s and N 1s features as those of the GOx electrolyte, but with additional C
1s feature at 283.3 eV and N 1s feature at 397.3 eV. The similar relative intensities of the
common C 1s and N 1s features for the GOx electrolyte to those found for FeꢀGOx
electrolyte indicate that the GOx molecules interact with Fe without dissociation. The
observed additional features for the FeꢀGOx electrolyte are found at the same binding energy
locations as the respective features in the FeꢀGOx HNPs, which indicates that the HNPs
contain FeꢀGOx complexes as their building blocks (before nanocrystallite formation). The
differences in the relative intensities of the C 1s and N 1s features for FeꢀGOx HNPs from
those for the dropꢀcast film of FeꢀGOx electrolyte suggest that there are more GOx
interactions with Fe involving the NHC=O group and C–O group in the HNPs than the dropꢀ
cast film.
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Figure 5: XPS spectra of N 1s and C 1s regions for dropꢀcast films of pristine GOx and of
FeꢀGOx electrolytes, and for FeꢀGOx hybrid nanoparticles (HNPs), all after 290 s sputtering.
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In order to investigate the biocompatibility of the FeꢀGOx HNPs, we studied the
effect of HNPs on cell proliferation. To be safe for use in the human body, Fe oxides usually
need to be coated with a biocompatible shell. These shells are mainly polymeric materials
that should be water soluble and nonꢀcytotoxic. However, in addition to adding an extra step
to the synthesis, the main drawback of polymeric coating is their poor longꢀterm chemical
stability of the inorganic core.48–51 We therefore have not used any coating in the present
experiment. In particular, we used the FeꢀGOx NPs directly deposited on an ITOꢀglass
substrate and evaluated their effects on HeLa cells. In the present experiment, we employed
ITOꢀglass instead of HꢀSi as the conductive substrate in order to obtain optical images of the
samples. It should be noted that the respective SEM images show no difference in the
morphologies of HNPs deposited on ITOꢀglass and HꢀSi substrates. HeLa cells were seeded
on ITOꢀglass substrates with the preꢀdeposited FeꢀGOx HNPs, and cell growth was then
monitored by brightꢀfield optical microscopy for 5 days (Figure 6a). Some parts of the
samples were scratched to remove the HNP cell seeding, and these were used as the control
so as to allow comparison with the areas with HNPs. As shown in Figure 6b, cell seeding
density, morphology, and growth appear to be similar in the bright regions (without HNPs)
and in the darker areas (with HNPs). In other words, the cells are able to grow on the
substrates coated with the FeꢀGOx HNPs as on substrates without the FeꢀGOx HNPs.
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Furthermore, in order to metabolically quantify the amount of active cells after
exposure to FeꢀGOx HNPs, we performed MTT (3ꢀ[4,5ꢀdimethylꢀ2ꢀthiazol]ꢀ2,5ꢀdiphenylꢀ
2Hꢀtetrazolium bromide) reduction experiments involving more delicate cells. Human
hepatoma cells (Liver, PLC/PRF/5, ATCC No: CRLꢀ8024) were cultured in Eagle’s
Minimum Essential Medium (EMEM) American Type Culture Collection (ATCC) with 10%
fetal bovine serum and 1% penicillinꢀstreptomycin. The cells were maintained in 5% CO2 in
humidity at 37 ºC. Before incubating the cells on the substrates, each substrate has been
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sterilized by first immersing in 70% ethanol thrice (10 minutes each), followed by PBS rinse.
The substrates were subsequently irradiated with UV light for 2 hours before use. Liver cells
(10,000 cells) were then seeded onto each substrate in a 12ꢀwell plate and incubated for 24
hours at 37 ºC. We employed the procedure for the MTT assay as outlined by the supplier
(Sigma/Aldrich). Figure 6c depicts the results from the MTT assay, which has been
performed in triplicates. Our results show quantitatively the cell population and activity on
the three substrates coated with FeꢀGOx HNPs compared to that on a plain ITOꢀglass used as
the control. Only a slight difference is observed, indicating that these FeꢀGOx HNPs are cellꢀ
safe and have no obvious detrimental effect on viability and cell growth.
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Figure 6d shows the magnetization curve of the FeꢀGOx HNPs (with an average size
of 400 nm) as determined by using a VSM. Evidently, the lack of hysteresis, along with the
observed 90 emu/g saturation magnetization, indicates that the FeꢀGOx HNPs exhibit
superparamagetic behavior. Due to the metallic nature of Fe in FeꢀGOx HNPs, the saturation
magnetization has a much larger value than the reported values for superparamagnetic iron
oxide NPs in the literature.52,53 The high saturation magnetization is especially important for
NPs when they are used as a contrast agent for MRI application. It should be noted that the
size of the superparamagnetic NPs cannot exceed 10 nm for Fe oxide and 20 nm for Fe.54–56
However, for inꢀvivo applications, magnetic NPs should have an appropriate size of 10ꢀ100
nm to maintain longer circulation times, because NPs smaller than 10 nm are removed by
renal clearance.57 For this reason, they are usually coated with a (polymer or organic) shell to
obtain the desired size.52 The present superparamagnetic FeꢀGOx HNPs are therefore quite
remarkable because the size of superparamagnetic HNPs can be easily controlled during the
coꢀelectrodeposition, thereby allowing us to eliminate the extra coating step. Since the
present HNPs are much larger than the superparamagnetic NP size regime, the observed
behavior supports our structural model shown schematically in Figure 2g. In other words, if
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the nanograins were just surfaceꢀfunctionalized by the GOx (i.e., without any hybridization),
then the nanocrystallites inside a nanograin are not isolated from one another by the GOx. In
this case, the NP should exhibit ferromagnetic and not the observed superparamagnetic
behaviour.
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Figure 6: Optical images of HeLa cells grown on ITO–glass substrate coated with FeꢀGOx
hybrid nanoparticles (HNPs), 400 nm in average size, after 5 days: (a) without and (b) with
sample scratching. (c) MTT data quantifying consistently high viability of human hepatoma
cells after 5 days of culturing on three separate ITOꢀglass substrates coated with FeꢀGOx
HNPs in relative to control (cultured on a plain ITOꢀglass substrate). (d) Magnetization curve
of the FeꢀGOx HNPs (with an average size of 400 nm).
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To demonstrate the general applicability of the present approach to other
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biomolecules even with different sizes and the application of HNPs to targeted drug delivery,
we prepared Feꢀp53p HNPs at 4 °C by coꢀelectrodeposition of Fe and p53p on a Hꢀ
terminated Si(100) substrate in a threeꢀelectrode cell. (It should be noted that p53p is
approximately 100 times smaller than GOx.) The electrolyte solution consisted of 5 mM
FeCl2 and 75 ꢁM p53p and 200 mM boric acid. The pH was adjusted to 6.5 before adding
p53p to the solution. Deposition was carried out at –1.2 V vs Ag/AgCl for different amounts
of deposition time. Figure 7a shows the SEM image of typical Feꢀp53p HNPs after 600 s
deposition. The corresponding XRD pattern (Figure 7b) shows metallic Fe with an average
grain size of 22 nm, which accounts for the observed rough surfaces of HNPs in the SEM
image. Similar to FeꢀGOx HNPs, these Feꢀp53p HNPs also exhibit superparamagnetic
behaviour with 60 emu/g saturation magnetization and no hysteresis (Figure 7c). The depthꢀ
profiling XPS spectra for the N 1s and C 1s regions in Figure 7d indicate the presence of N–
Fe and C–Fe features at 397.3 eV and 283.3 eV, respectively. The emergence of these
features only upon longer sputtering time (1400 s) confirms the intermixing of Fe with p53p
in the core (i.e. subꢀsurface) region and the hybrid nature of NPs. To verify that the p53p in
the HNP does not change after hybridization and preserves its bioactivity, we examine p53pꢀ
Mdm2 bonding ability. For this purpose, we incubated pristine Fe NPs and Feꢀp53p HNPs in
the solution of Mdm2ꢀGST for 4 hours. We then washed both samples thoroughly with
deionized water. The SEM images obtained after incubation show extra materials on top of
Feꢀp53p HNPs but not on pristine Fe NPs, which confirms the attachment of Mdm2 to p53p
and therefore the bioactivity of p53p in the HNP (Supporting information, Figure S6).
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Figure 7: (a) SEM images (scale bars are 200 nm), (b) XRD pattern, (c) hysteresis loop, (d)
depthꢀprofiling XPS spectra of N 1s and C 1s regions of Feꢀp53p HNPs obtained after 600 s
deposition.
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In summary, FeꢀGOx and Feꢀp53p HNPs have been successfully obtained by
electrodeposition of FeꢀGOx and Feꢀp53p complexes directly as the building blocks of the
nanograins at 4 °C. We propose a “stoneꢀandꢀmortar” architecture for the HNP, in which
GOx or p53p molecules behave as the mortar to keep the Fe nanocrystallites together. HIM
and FIBꢀSEM multi crossꢀsectional studies both show the nanograins interconnected to one
another by the GOx or p53p molecules in the HNPs. Furthermore, both TOFꢀSIMS and
depthꢀprofiling XPS results further confirm the intermixed nature of the Feꢀbiomolecule NPs,
in which the biomolecules are incorporated throughout the HNP without affecting their
bioactivity. Of special interest is that hybridization of FeꢀGOx and Feꢀp53p HNPs is
obtained by complex formation of Fe with the biomolecule (GOx or p53p) before deposition
onto the substrate and the growth is maintained by these Feꢀbiomolecule complexes as the
building blocks. Furthermore, these HNPs are found to be superparamagnetic and could be
made in the desirable size regime. As demonstrated by their apparent lack of any detrimental
effect on cell growth, their biocompatibility, together with their strong magnetic properties,
make these HNPs promising agents for MRI and targeted drug delivery systems. These
HNPs can be easily harvested from the substrate and dispersed in an aqueous solution by
sonication. Incorporating two different biomolecules with very different sizes confirms the
general applicability of the present synthetic approach. The present method of incorporating
bioactive molecules into magnetic (and nonꢀmagnetic) nanostructures is therefore not limited
to GOx or p53p. Hybridization of GOx as a waterꢀsoluble enzyme and p53p as an anticancer
peptide with magnetic nanostructures therefore offers an effective immobilization method to
enable them for use not only as reusable sensors and magnetic contrast agents, but also in
greatly improved drug delivery and recovery systems.
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Page 29 of 35
Journal of the American Chemical Society
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Supporting Information Available
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Experimental details, UV and CV data, SEM images, TOFꢀSIMS mapping and depth profile.
This information is available free of charge via the internet at http://pubs.acs.org.
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Acknowledgement
This work was supported by the Natural Sciences and Engineering Research Council of
Canada.
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