Low temperature preparation of a graphene-cobalt microsphere hybrid by borohydride-initiated reduction for enriching proteins and peptides

Article abstract of DOI:10.1039/c4tb00302k

A graphene-cobalt microsphere hybrid was prepared by the chemical reduction of a mixture containing graphene oxide and cobalt chloride. It was found that a little amount of potassium borohydride or sodium borohydride could initiate the hydrazine-reduction of the mixture at a low temperature of 80 °C. The structure of the material was investigated by scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, vibrating sample magnetometery, Fourier transform infrared spectroscopy, Raman spectroscopy, and Brunauer-Emmett-Teller (BET) techniques. The average size of the cobalt microspheres on graphene sheets was measured to be ～590 nm. Magnetic investigations indicated that the graphene-cobalt microsphere hybrid exhibited ferromagnetic behavior at room temperature. In addition, the magnetic hybrid was successfully employed in the enrichment and identification of low-abundance proteins and peptides in combination with mass spectrometry. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4tb00302k

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Materials Chemistry B
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Low temperature preparation of a graphene–
cobalt microsphere hybrid by borohydride-initiated
Cite this: J. Mater. Chem. B, 2014, 2,
reduction for enriching proteins and peptides†
5220
*
Huimin Ge, Huimin Bao, Luyan Zhang and Gang Chen
A graphene–cobalt microsphere hybrid was prepared by the chemical reduction of a mixture containing
graphene oxide and cobalt chloride. It was found that a little amount of potassium borohydride or
sodium borohydride could initiate the hydrazine-reduction of the mixture at a low temperature of 80 ꢀC.
The structure of the material was investigated by scanning electron microscopy, transmission electron
microscopy, energy dispersive spectroscopy, vibrating sample magnetometery, Fourier transform
infrared spectroscopy, Raman spectroscopy, and Brunauer–Emmett–Teller (BET) techniques. The
average size of the cobalt microspheres on graphene sheets was measured to be ꢁ590 nm. Magnetic
investigations indicated that the graphene–cobalt microsphere hybrid exhibited ferromagnetic behavior
at room temperature. In addition, the magnetic hybrid was successfully employed in the enrichment and
identification of low-abundance proteins and peptides in combination with mass spectrometry.
Received 23rd February 2014
Accepted 16th June 2014
DOI: 10.1039/c4tb00302k
www.rsc.org/MaterialsB
Ag,13 Pt,14 ZnS,15 MnO2,16 NiO,17 etc. In the past few decades,
various approaches have been developed for the preparation of
1. Introduction
cobalt particles owing to their unusual magnetic properties and
industrial importance.18,19 Because exfoliated graphene sheets
have a large specic surface area (2600 m2 gÀ1 (ref. 20)) and
unique properties, it is interesting to deposit cobalt particles on
them to prepare magnetic hybrids. To date, graphene–cobalt
hybrids are mainly prepared by high-temperature hydrogen
reduction,21,22 hydrothermal treatment,23,24 solvothermal
method,25,26 sol–gel autocombustion,27 ame spray pyrolysis,28
chemical vapor deposition,29 mechanical ball milling,30 and
electrodeposition.31 Besides the last two methods, other
approaches need to be performed at high temperatures in the
range of 180 to 800 ꢀC. It is of high importance to develop
efficient approaches for the rapid preparation of the magnetic
graphene–cobalt hybrid in aqueous solution at low
temperatures.
Proteomics is the large-scale study of proteins, particularly
their structures and functions. As one of the fastest developing
areas in biological research, proteomics has drawn more and
more attention.32,33 One of its most important tasks is to develop
efficient and rapid approaches to identify various proteins.
Peptide mapping is a commonly used strategy for protein
identication. Proteins are usually digested into peptides that
are subsequently identied by matrix-assisted laser desorption/
ionization time-of-ight mass spectrometry (MALDI-TOF MS)
along with database searching.34 Although MALDI-TOF MS is
highly sensitive, it is usually insufficient for the detection of
low-abundance peptides or proteins isolated from biological
samples. Prior to MS analysis, a prior enrichment of the
samples is usually inevitable. Because magnetic nanomaterials
As an important allotrope of carbon, graphene has a two-
dimensional nanostructure of sp2-bonded carbon atoms that
are arranged in a honeycomb pattern.1,2 Since it was successfully
isolated and characterized by Novoselov and Geim in 2004,
graphene has received ever-increasing attention and become a
rapidly rising star in the elds of materials sciences and
condensed-matter physics owing to its unique two-dimensional
structure and excellent physical and chemical properties.3–9
Nowadays, graphene is mainly prepared by chemical reduc-
tion of graphene oxide (GO), chemical vapor deposition from
carbon precursors, and exfoliation of graphite by a variety of
approaches.10 Among them, the rst approach is commonly
used to produce graphene at low cost. Because GO sheets bear a
great deal of oxygen-containing groups, they can be well
dispersed in aqueous solution for the preparation of graphene-
based hybrids.11 When GO sheets are reduced to hydrophobic
graphene sheets in aqueous solution, they tend to aggregate to
form graphitic structures under the drive of the interlayer p–p
interaction, van der Waals force, and hydrophobic interaction.
To solve the problem, a variety of inorganic particles are deco-
rated on graphene sheets to enlarge the interplanar spacing
between them. A series of graphene-based hybrids have been
prepared by decorating them with the particles of Cu,8 Au,12
School of Pharmacy, Department of Chemistry, Fudan University, 826 Zhangheng
Road, Shanghai 201203, China. E-mail: gangchen@fudan.edu.cn; Fax: +86-21-
51980168; Tel: +86-21-51980061
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4tb00302k
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ꢀ
can be easily manipulated with the aid of external magnetic allowed to proceed in a 35 C water bath for 30 min, 92 mL of
elds, they indicate great promise for the enrichment of doubly distilled water was gradually added. The temperature of
proteins and peptides in proteomics research.34–36 A variety of the mixture was kept at 98 ꢀC for 40 min to increase the
novel magnetic nanomaterials were synthesized by assembling oxidation degree of the product. And then, the volume of the
various materials on magnetic iron oxide microspheres fol- resultant suspension was adjusted to 280 mL with doubly
lowed by surface modication.34–36 Recently, graphene nano- distilled water. Aer 6 mL of hydrogen peroxide solution (30%
sheets were covalently attached on silica particles37 and w/w) was added, the color of the suspension changed from
magnetic iron oxide spheres38 for the enrichment of proteins brown to bright-yellow. The prepared oxidized graphite could be
and peptides. However, to the best of our knowledge, no easily isolated from the solution by vacuum ltration. The
adsorbent composed of metallic cobalt and graphene sheets has sulfuric acid and salt impurities in the crude product were
been reported for enriching peptides and proteins.
removed by washing with 5% (w/w) hydrochloric acid and
In this work, a graphene–cobalt microsphere (CoMS) hybrid doubly distilled water with the aid of vacuum ltration. The wet
ꢀ
was prepared by the chemical reduction of GO sheets and product was dewatered by vacuum drying (50 C).
cobalt(II) chloride in a hydrazine-containing solution at a lower
temperature of 80 ꢀC. It was demonstrated that a little amount
2.3. Preparation of the graphene–CoMS hybrid
of potassium borohydride or sodium borohydride could initiate
To prepare the graphene–CoMS hybrid, 0.3 g oxidized graphite
the reduction reaction and the reduction time was reduced to as
powder was dispersed in 100 mL doubly distilled water and
short as 60 min. In addition, the prepared hybrid was employed
sonicated in an ultrasonic cleaner (SKQ-2200, frequency 56 kHz,
in the enrichment and identication of low-abundance proteins
100 W) for 1 h to exfoliate oxidized graphite particles to GO
and peptides in combination with MALDI-TOF MS. It exhibits
sheets. Aer an aqueous solution (5 mL) of CoCl2$6H2O (1.19 g,
excellent magnetic responsibility and enrichment capability. In
5 mmol) was added, the mixture was sonicated for 10 min. And
the prepared hybrid, hydrophobic graphene nanosheets with
then, 5 mL of hydrazine hydrate (85% w/w) and 10 mL of
high specic surface area can capture the proteins and peptides
ammonium hydroxide solution (the content of NH3, 25–28% w/
in solution while its cobalt component facilitates rapid
w) were successively added into the mixture that was vigorously
magnetic separation. The MALDI-TOF-MS signals of low-abun-
stirred. The mixture solution was then heated in an 80 ꢀC water
dance proteins and peptides are substantially enhanced aer
bath for 10 min. To initiate the hydrazine-based reduction
enriching with the unique magnetic hybrid. The preparation
reaction, 20 mg potassium borohydride (or 15 mg sodium
details, characterization, advantages, and application of the
borohydride) was directly dissolved in the mixture with the aid
graphene–CoMS hybrid are reported in the following sections.
of mechanical agitation. It could be observed that the color of
the mixture immediately turned from brown to black while a
great deal of colorless gas released from the solution. The
2. Experimental
2.1. Reagents and solutions
reduction reaction was allowed to proceed at 80 ꢀC for 60 min.
The obtained graphene–CoMS hybrid could be easily isolated
Cobalt(II) chloride hexahydrate (CoCl2$6H2O), hydrazine
hydrate (85% w/w), potassium borohydride, sodium borohy-
dride, graphite powder, potassium permanganate, sodium
nitrate, ammonium hydroxide solution (the content of NH3,
25–28% w/w), hydrogen peroxide solution (30% w/w), and
sulfuric acid (98% w/w) were all supplied by SinoPharm
(Shanghai, China). Cytochrome c (Cyt-c) from horse heart,
lysozyme (LYZ) from chicken egg, bovine hemoglobin (HEM),
trypsin from bovine pancreas, triuoroacetic acid (TFA), aceto-
nitrile (ACN) and a-cyano-4-hydroxycinnamic acid (CHCA) were
supplied by Sigma (St. Louis, MO, USA). The stock solutions of
Cyt-c, LYZ, and HEM (1 mg mLÀ1) were prepared in 10 mM
NH4HCO3 solution (pH 8.1) and were denatured in a 95 ꢀC water
bath for 15 min.
form the solution by vacuum ltration and was puried by
washing with copious amounts of doubly distilled water.
Finally, it was washed with absolute ethanol and dried in a
vacuum. Based on the thermogravimetric analysis (TGA) of the
hybrid, the weight ratio of metallic cobalt to graphene within
the hybrid was estimated to be approximately 2 : 1.
CoMSs were also prepared by mixing an aqueous solution
(20 mL) of CoCl2$6H2O (1.19 g, 10 mmol) with the solution
mixture of 3 mL of hydrazine hydrate (85% w/w) and 10 mL of
ammonium hydroxide solution (the content of NH3, 25–28%
ꢀ
w/w). The prepared mixture was heated in an 80 C water bath
for 10 min. And then, 20 mg potassium borohydride was dis-
solved in the mixture to initiate the reduction reaction. Aer
30 min, CoMSs could be easily separated from the solution with
the aid of an NdFeB magnet (ꢁ3000 Gs). Graphene was
prepared by the reduction of GO sheets with hydrazine hydrate
as a reducing agent.8
2.2. Preparation of oxidized graphite
Oxidized graphite was synthesized from graphite by a modied
Hummers method.12 Briey, 2 g graphite powder was dispersed
in 46 mL of sulfuric acid (98% w/w) under agitation. Subse-
2.4. Apparatus
quently, 1.2 g sodium nitrate and 6 g potassium permanganate The surface morphologies were studied and energy dispersive
were successively added into the mixture in an ice bath. Note spectroscopy (EDS) spectra of the prepared materials were
that both compounds should be added slowly to prevent the recorded by using a scanning electron microscope (PHILIPS XL
temperature from exceeding 20 ꢀC. Aer the reaction was 30, Eindhoven, The Netherlands). Transmission electron
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microscopy (TEM) images were observed using a JEOL model each sample solution was mixed with 20 mL of 10 mg mLÀ1
JEM-2011(HR) transmission electron microscope (JEOL Ltd., hybrid suspension in a 1.5 mL centrifuge tube that was vibrated
Japan) at 200 kV. X-ray diffraction (XRD) measurements were for 5 min at ꢁ25 ꢀC. The peptide-captured hybrid was separated
carried out using a Rigaku D/max-rB diffractometer (Rigaku, and rinsed with water with the aid of a magnet. The adsorbed
Tokyo, Japan) with CuK-a1 radiation (40 kV, 60 mA). A vibrating- peptides could be extracted with 10 mL of 50% aqueous ACN
sample magnetometer (Model 155, EG&G Princeton Applied containing 0.1% TFA with the aid of the magnet for subsequent
Research, USA) was used at room temperature to study the MS analysis.
magnetic properties of the magnetic materials. TGA was per-
formed by using a PerkinElmer Pyris 1 DTA-TGA instrument in
2.7. MALDI-TOF-MS analysis
air at a heating rate of 10 ꢀC minÀ1. Raman spectra were
Prior to MALDI-TOF-MS analysis, each sample (0.5 mL) was
acquired on a Labram-1B (Dilor, France) confocal microscopy
Raman spectrometer with a 632.8 nm wavelength incident laser
light. The Fourier transform infrared (FT-IR) spectra of GO,
graphene, CoMS, and graphene–CoMS hybrid were recorded
was operated at an accelerating voltage of 20 kV. A 1000 Hz
using a FT-IR spectrometer (NEXUS470, NICOLET). Nitrogen
spotted on a MALDI plate. Aer the solvent was evaporated, 0.5
mL of matrix solution (50% aqueous ACN containing 4 mg mLÀ1
CHCA and 0.1% TFA) was dropped on it. The MS instrument
pulsed Nd:YAG laser at 355 nm was used. Prior to use, the MS
adsorption–desorption isotherms were measured with a Quad-
instrument was calibrated with the tryptic digest of myoglobin
raSorb SI surface area and pore size analyzer (Quantachrome
in an internal calibration mode. Reector mode was used to
Instruments, Florida, USA) at 77 K. The specic surface area of
detect low-mass peptides while high-mass proteins were
the prepared materials was calculated by the Brunauer–
measured in a linear mode. GPS Explorer soware from Applied
Emmett–Teller (BET) method in the relative pressure range of
Biosystems with Mascot as a search engine and SwissProt as a
0.03 to 0.30. The pore size distribution of the materials was
database was used to identify proteins based on the recorded
peptide mass ngerprinting (PMF) spectra of the protein
calculated by the Barrett–Joyner–Halenda (BJH) approach based
on the desorption branches of the isotherms. Prior to the
samples. The search was performed based on the monoisotopic
measurements, all samples were degassed at 250 ꢀC under
vacuum for 3 h. All MALDI-TOF-MS measurements were per-
formed in positive ion mode using a 5800 proteomics analyzer
MH+ mass values of peptides while the missed cleavages of
peptides were allowed up to 1.
(Applied Biosystems, Framingham, MA).
3. Results and discussion
2.5. Enrichment of proteins by the graphene–CoMS hybrid
Because GO sheets have a great amount of negatively charged
An accurate amount (100 mg) of the prepared graphene–CoMS
carboxylic groups on their surface,11 Co2+ tends to be attracted
hybrid was dispersed in 10 mL doubly distilled water with the
on them by electrostatic interactions. The GO sheets bearing
aid of sonication. The suspension of the magnetic hybrid or
Co(II) ions can be reduced by using suitable reducing agents to
pristine CoMSs (10 mg mLÀ1, 20 mL) was added to 1 mL of
form the magnetic graphene–CoMS hybrid. In this work,
mixture solution of Cyt-c and LYZ (1 ng mLÀ1 each) in a poly-
potassium borohydride or sodium borohydride was employed
propylene centrifuge tube. Subsequently, the mixture was
to initiate the chemical reduction of GO sheets and cobalt ions
shaken for 5 min at ꢁ25 ꢀC. An NdFeB magnet was used to
in a hydrazine-containing aqueous solution. Aer the mixture
attract the magnetic hybrid to the bottom of the vial. Aer the
was heated at 80 ꢀC for 60 min, the magnetic black hybrid was
clear supernatant was decanted, the protein-captured hybrid
separated for the following characterization and enrichment of
was rinsed with water twice and redispersed in 10 mL of 50%
peptides and proteins. When 4 mg of the prepared hybrid was
aqueous ACN containing 0.1% TFA with the aid of the magnet.
dispersed in 4 mL of water, it could remain in the suspension
The eluent containing enriched proteins was collected for
for at least 1 h without visible sediment (the le inset in Fig. 1A).
subsequent MS analysis.
The dispersed hybrid could be separated to the wall within
1 min with the aid of an NdFeB magnet (ꢁ3000 Gs) (the right
2.6. Enrichment of peptides by the graphene–CoMS hybrid
inset in Fig. 1A). The redispersion and separation processes
HEM and Cyt-c were digested into peptides following a previ- could be repeated readily. The high dispersibility and sensitivity
ously reported procedure.39 Briey, the stock solutions of to a magnetic eld exhibited by the hybrid indicates great
trypsin (1 mg mLÀ1) were prepared in 50 mM acetic acid promise for the convenient enrichment of proteins and
aqueous solution. For tryptic digestion, the stock solution of peptides.
HEM and Cyt-c (200 mL) was mixed with 5 mL trypsin solution
A vibrating sample magnetometer was used to record the
and 795 mL of 10 mM NH4HCO3 solution (pH 8.1) and incubated hysteresis loops of the prepared graphene–CoMS hybrid at
at 37 ꢀC for 18 h. The weight ratio of trypsin to protein substrate room temperature to characterize its magnetic properties
was 1 : 40. An accurate volume (25 mL) of each digest was further (Fig. 1A). The saturation magnetizations (Ms), coercivities (Hc)
diluted with doubly distilled water to 10 mL to prepare the and remanent magnetizations (Mr) of the hybrid are determined
sample solution (0.5 ng mLÀ1 for each protein) for subsequent to be 80.74 emu gÀ1, 377.1 Oe, and 10.31 emu gÀ1, respectively.
enrichment and MALDI-TOF-MS analysis. To demonstrate the The presence of Hc and Mr in the hysteresis loops indicates
enrichment capability of the graphene–CoMS hybrid, 1 mL of that the hybrid exhibits ferromagnetic behavior. The
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graphene sheets, a crumpled silk wave-like morphology is
observed in the SEM and TEM images of the hybrid (Fig. 2d, e
and S1a†). Monodispersed CoMSs are individually decorated on
the surface of graphene nanosheets. As illustrated in Fig. 2f, the
average size of the CoMSs on graphene sheets is estimated to be
ꢁ590 nm. CoMSs are well dispersed and embedded throughout
the graphene matrix. The SEM image of pristine CoMSs indi-
cates that the size of most CoMSs is in the range of 200 to 400
nm with the average size of ꢁ320 nm (Fig. 2a–c). In comparison
with pristine CoMSs, the aggregation of CoMSs in the gra-
phene–CoMS hybrid is signicantly reduced owing to their
interactions with graphene sheets and the steric hindrance of
graphene sheets. Although the hybrid had been sonicated in
ethanol before TEM measurements, some CoMSs are found on
the surface of graphene sheets, indicating the strong interaction
between CoMSs and graphene nanosheets. During the prepa-
ration of the graphene–CoMS hybrid, GO sheets played roles of
a dispersant, a two-dimensional growth template for CoMSs,
and a precursor of graphene.
The EDS spectrum of the prepared graphene–CoMS hybrid
was recorded to investigate the element constituents (Fig. S1b†).
Because a layer of gold was sputtered on it to record the SEM
image, the peaks of gold were also found. The results indicate
that the graphene–CoMS hybrid is mainly composed of cobalt,
carbon and oxygen elements. Based on the fact that no peak of
cobalt hydroxide is observed in the XRD patterns of the hybrid
(Fig. S2b†), the oxygen element in the material mainly comes
from the residual oxygen-containing functional groups (such as
epoxy and hydroxyl groups) on the graphene sheets as well as
the oxygen and water molecule absorbed in the hybrid.
The FT-IR spectra of graphene, graphene–CoMS hybrid,
graphene–CoMS hybrid, CoMSs, and GO are illustrated in
Fig. S3.† As illustrated in Fig. S3d,† absorption bands of GO are
observed at 3412, 1730, and 1053 cmÀl, which are attributed to
the stretching vibrations of O–H, C]O, and C–O–C, respec-
tively. The peaks at 1624, 1407 and 1224 cmÀl correspond to the
vibration of carboxyl groups.15 Fig. S3a and S3b† indicate that
these peaks become weak or disappear when GO is reduced to
graphene by hydrazine. The FT-IR spectrum of the graphene–
CoMS hybrid is similar to that of graphene although the peaks
of CoMSs below 750 cmÀl can be observed, implying that the
amount of hydroxyl, carbonyl and carboxyl groups in graphene
and the graphene–CoMS hybrid substantially decreases aer
chemical reduction.
Fig. 1 (A) Hysteresis loops of the graphene–CoMS hybrid and (B and
C) the magnetic response of the 1 mg mLÀ1 hybrid prepared by
reducing the mixture of GO sheets and cobalt(II) chloride in a hydra-
zine-containing aqueous solution at 90 ꢀC for 60 (B) and 240 (C) min
in the absence of potassium borohydride. Also shown in the insets of
Fig. 1A are photographs illustrating the dispersion (left inset) and
magnetic separation (right inset) processes of a suspension of the 1 mg
mLÀ1 graphene–CoMS hybrid in water. (a) Graphene–CoMS hybrid
suspension in water; (b) glass vial; (c) water; (d) NdFeB magnet
(ꢁ3000 Gs).
ferromagnetism of the graphene–CoMS hybrid can be attrib-
uted to its CoMS component. The saturation magnetization of
the CoMSs in the hybrid is calculated to be 121.11 emu gÀ1
based on the content of cobalt in the hybrid (2/3, w/w). It is
lower than the reported value of bulk cobalt (168 emu gÀ1
)
40
owing to the small particle size and the inevitable surface
oxidation of CoMSs in the hybrid.41
Thermogravimetry was employed to evaluate the weight
fraction of graphene in the prepared hybrid. Fig. S3e and S3f†
show the TGA curves of graphene and the graphene–CoMS
hybrid in air. The weight of the hybrid starts to increase at
300 ꢀC owing to the oxidation of CoMSs in the hybrid. In
comparison with the TGA curves of pristine graphene, the
obvious weight loss of the hybrid in the temperature range of
470 to 580 ꢀC can be attributed to the decomposition and
combustion of graphene. When the hybrid is heated to above
For comparison, hydrazine was also employed to reduce the
mixture of GO sheets and cobalt(II) chloride in a hydrazine-
containing aqueous solution at 90 C in the absence of potas-
ꢀ
sium borohydride. Other conditions were the same as those for
borohydride-initiated reduction. As illustrated in Fig. 1B and C,
the prepared hybrids exhibited no obvious response when the
NdFeB magnet was placed close beside the vial for 30 min.
Obviously, borohydride could imitate the formation of
the magnetic graphene–CoMS hybrid at a lower temperature
ꢀ
580 C in air, its graphene component burns completely while
the metallic cobalt in it is oxidized to form Co3O4.42 Based on
the TGA curve of the hybrid and the molecular weight of Co3O4,
ꢀ
of 80 C.
The morphology of the prepared graphene–CoMS hybrid was
examined by SEM and TEM. As a characteristic prole of
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Fig. 2 SEM images of (a and b) CoMSs and (d and e) graphene–CoMS hybrid and particle size distribution of (c) pristine CoMSs and (f) CoMSs on
graphene sheets (the number of particles counted, 200). Magnification; (a and d) Â5000 and (b and e) Â10 000.
the mass ratio of metallic cobalt to graphene within the hybrid disordered carbon atoms.46 The intensity ratio of the D to the G
can be estimated to be approximately 2 : 1.
band (ID/IG) is related to the average size of sp2 carbon
Fig. S2† illustrates the XRD patterns of graphene and the domains.47 Aer chemical reduction of GO sheets, the size of
graphene–CoMS hybrid. Diffraction peaks assigned to graphene the reestablished conjugated sp2 carbon domains is usually
at 24.2ꢀ (corresponding to the indices of (002))43 can be clearly smaller than that of the original graphite layer, which results in
seen for both graphene and the graphene–CoMS hybrid, indi- the increase of the ID/IG ratio.48 Such a change is also observed
cating that the graphene structure was not destroyed aer in Fig. S4,† in which the ID/IG ratio of the graphene in the hybrid
chemical reduction. In the XRD pattern of the graphene–CoMS (1.62) is higher than that of GO (1.29). It has been demonstrated
hybrid, all the four characteristic diffraction peaks at 41.9ꢀ, that the increase in the ratio of D to G band intensity (ID/IG) can
44.7ꢀ, 47.7ꢀ and 75.1ꢀ can be attributed to the (111), (200) and be attributed to an increase in the number of isolated sp2
(220) crystalline planes of the hexagonal cobalt (JCPDS 05-0727). domains.47,49 Meanwhile, the D and G bands in the Raman
The results are good in agreement with the data in a previous spectrum of the hybrid shi toward lower frequency slightly,
report.44 It can be concluded that the hybrid prepared by the which indicates that GO is reduced to form graphene in the
in situ chemical reduction of GO sheets and Co(II) ions is a hybrid as the layer thickness increases.50
hybrid of graphene and cobalt and no characteristic peaks of
In addition, the nitrogen adsorption–desorption isotherms
Co(OH)2 is observed. It has been reported that cobalt nano- (Fig. 3) and pore size distribution (Fig. S5†) of graphene, gra-
particles are readily oxidized to paramagnetic Co2O3 nano- phene–CoMS hybrid and CoMSs were measured to investigate
particles (average size, ꢁ20 nm).45 However, both the results their surface properties. Obviously, the isotherms of the three
TGA and XRD for the hybrid indicate that the cobalt element in materials can be assorted as type IV according to the IUPAC
it mainly exists in the form of zero-valent cobalt, which implies classication. The existence of distinct hysteresis loops indi-
that the oxidation of CoMSs in the hybrid is not pronounced. cates that they possess nanoporous structures.51 Table 1
The size of the CoMSs in the hybrid is in the range of 400 to
800 nm, which is much higher than that of the reported cobalt
nanoparticles (less than 20 nm). The specic surface area of the
CoMSs prepared in this work is lower so that the oxidation
degree is signicantly reduced. More importantly, graphene
sheets may protect the CoMSs in the hybrid from oxidation
because CoMSs are embedded throughout graphene matrix
(Fig. 2d and e).
In this work, Raman spectroscopy was employed to charac-
terize the ordered and disordered crystal structures of graphene
in the hybrid. Two Raman peaks at around 1560 and 1360 cmÀ1
,
which correspond to G and D bands, were observed in the
Raman spectra of GO and the graphene–CoMS hybrid (Fig. S4†).
The G band can be attributed to the bond stretching of all pairs
of sp2 bonded carbon atoms while the D band is related to the
Fig. 3 Nitrogen adsorption–desorption isotherms of (a) graphene, (b)
GR–CoMS hybrid, and (c) CoMSs at 77 K.
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Table 1 Porous structure parameters of graphene, graphene–CoMs hybrid, and CoMSs
Sample
BET surface area (m2 gÀ1
)
BJH cumulative pore volume (cm3 gÀ1
)
BJH pore diameter (nm)
Graphene
Graphene–CoMS hybrid
CoMSs
316.94
89.53
17.88
0.4738
0.3314
0.0911
3.83
3.84
2.36
summarizes the BET surface area, BJH cumulative pore volume
and BJH pore diameter of graphene, graphene–CoMS hybrid
and CoMSs. The BET surface area of the graphene–CoMS hybrid
(89.53 m2 gÀ1) is much higher than that of CoMSs (17.88), which
is important for the enrichment of proteins and peptides.
However, it is lower than that of graphene (316.94 m2 gÀ1). The
BJH pore size distribution of the three samples is calculated and
shown in Fig. S5.† The pore size of graphene is mainly in the
range of 2.0 to 4.5 nm. However, the pore size range of the
graphene–CoMS hybrid is broadened to the range of 1.4 to
70.0 nm owing to the presence of CoMSs. Although the most
probable pore size of the graphene–CoMS hybrid (3.84 nm) is
similar to that of graphene (3.83 nm), it is higher than that of
CoMSs (2.36 nm). The BJH cumulative pore volume of the gra-
phene–CoMS hybrid (0.3314 cm3 gÀ1) is almost 70% of the value
for pristine graphene (0.4738 cm3 gÀ1) although the mass
fraction of graphene in the hybrid is ꢁ33%. The results indicate
that CoMSs in the hybrid may enlarge the interplanar spacing
between graphene sheets.
The graphene–CoMS hybrid consists of magnetic CoMSs and
hydrophobic graphene sheets that have high specic surface
area, indicating great promise for the rapid enrichment of
peptides and proteins. In this work, it was dispersed in a
mixture solution containing Cyt-c and LYZ (1 ng mLÀ1 each) to
investigate its enrichment performance. As indicated in Fig. 4a,
the signals of the two proteins in the MALDI-TOF mass spec-
trum are very low prior to enrichment. The signal-to-noise (S/N)
ratios are measured to be 15.14 and 8.95 for Cyt-c and LYZ
(Fig. 4a). Aer the mixture is enriched by the magnetic hybrid,
the S/N ratios of Cyt-c and LYZ in the eluent signicantly
increase to 5192.31 and 2894.83 (Fig. 4b). Obviously, the signals
of both proteins are dramatically enhanced aer enrichment.
Both proteins can be easily identied based on the determined
molecular mass (12 362 Da for Cyt-c ([M + H]+) and 14 307 Da for
LYZ ([M + H]+)). The results indicate that the graphene–CoMS
hybrid exhibits satisfactory performance in enriching low-
abundance proteins.
To evaluate the enrichment capability of the graphene–
CoMS hybrid to peptides, it was employed to capture peptides in
the tryptic digests of HEM and Cyt-c (0.5 ng mLÀ1) for MS-based
peptide mapping and protein identication. As indicated in
Fig. 5a and c, only 1 or 2 peptides are detected in the digests
before enrichment and the proteins cannot be identied.
Fig. 5b and d show the mass spectra for the tryptic digests of
HEM and Cyt-c aer being enriched with the graphene–CoMS
hybrid. Clear peptide mass ngerprintings were obtained with
signicantly enhanced signals. All the identied peptides in
both digests are summarized in Tables 2 and 3. The numbers of
Fig. 4 MALDI-TOF mass spectra of a mixture containing Cyt-c and
LYZ (1 ng mLÀ1 each) (a) before and (b) after enrichment with the
graphene–CoMS hybrid.
the matched peptides and the amino-acid sequence coverages
are 14 and 90% for HEM and 12 and 91% for Cyt-c. In addition,
131 out of the 145 possible amino acids of HEM and 95 out of
the 105 possible amino acids of Cyt-c have been identied. The
results indicated that both protein samples are positively
identied aer being enriched by the graphene–CoMS hybrid,
implying that it is an ideal magnetic material for the convenient
and efficient enrichment of low-abundance proteins and
peptides.
In addition, pristine CoMSs were employed to enrich the
peptides of HEM and Cyt-c as a control experiment. Fig. S6†
illustrates the MALDI-TOF mass spectra of the tryptic digests of
HEM and Cyt-c (0.5 ng mLÀ1 each) aer being enriched with
pristine CoMSs. Only one peptide was found matched in the
PMF of HEM while no peak of tryptic peptides was observed in
the mass spectra for the digest of Cyt-c. Obviously, the high
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Fig. 5 MALDI-TOF mass spectra of the tryptic digests of (a and b) HEM and (c and d) Cyt-c (0.5 ng mLÀ1 each) before (a and c) and after (b and d)
enrichment with the graphene–CoMS hybrid. The peaks marked with asterisks represent the identified peptides in the tryptic digests of proteins.
Table 2 Peptide mapping results for the tryptic digests of 0.5 ng mLÀ1 HEM before and after enrichmenta
No.
Position
Peptide
Before enrichment
Aer enrichment
1
2
3
4
5
6
7
8
8–16
19–29
8–18
AAVTAFWGK
VDEVGGEALGR
AAVTAFWGKVK
LLGNVLVVVLAR
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
104–115
30–39
17–29
120–131
132–145
1–16
66–81
116–131
40–58
76–94
40–60
ꢂ
LLVVYPWTQR
VKVDEVGGEALGR
EFTPVLQADFQK
VVAGVANALAHRYH
MLTAEEKAAVTAFWGK
VLDSFSNGMKHLDDLK
NFGKEFTPVLQADFQK
FFESFGDLSTADAVMNNPK
HLDDLKGTFAALSELHCDK
FFESFGDLSTADAVMNNPKVK
ꢂ
ꢂ
ꢂ
ꢂ
9
ꢂ
10
11
12
13
14
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
Sequence coverage (%)
Peptides matched
Amino acids identied
15
22
2
90
14
131
a
The matched peptides are labeled with “ꢂ”.
enrichment capability of the graphene–CoMS hybrid can be
To test the stability of the prepared graphene–CoMS
attributed to its graphene component that has large surface hybrid, it was sealed in a polypropylene tube and stored in a
area, hydrophobic surface, high dispensability, and the unique glass desiccator containing blue silica gel beads at room
structure. Peptides and proteins in sample solutions can be temperature for 8 months. No obvious change in its weight
enriched on the surface of the hybrid via hydrophobic interac- and saturation magnetization was observed, implying that
tions, p–p interactions, and van der Waals force.37,38 The the stability of the hybrid was satisfactory under the storage
excellent ferromagnetic properties of the hybrid originate from conditions. More importantly, the hybrid still attained the
its cobalt component so that it can be easily separated from the ability to enrich proteins and peptides 8 months aer it was
solution.
synthesized.
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Table 3 Peptide mapping results for the tryptic digests of 0.5 ng mLÀ1 Cyt-c before and after enrichmenta
No.
Position
Peptide
Before enrichment
Aer enrichment
1
2
3
4
5
6
7
8
81–87
74–80
1–8
MIFAGIK
KYIPGTK
MGDVEKGK
MIFAGIKK
TGPNLHGLFGR
TGPNLHGLFGRK
TEREDLIAYLK
HKTGPNLHGLFGR
KTGQAPGFTYTDANK
IFVQKCAQCHTVEK
TGQAPGFTYTDANKNK
GITWKEETLMEYLENPK
ꢂ
ꢂ
ꢂ
81–88
29–39
29–40
90–100
27–39
40–54
10–23
41–56
57–73
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
9
ꢂ
10
11
12
ꢂ
ꢂ
ꢂ
Sequence coverage (%)
Peptides matched
Amino acids identied
10
1
11
91
12
95
a
The matched peptides are labeled with “ꢂ”.
5 X. Wang, J. Y. Li, W. D. Qu and G. Chen, J. Chromatogr. A,
2011, 1218, 5542.
4. Conclusions
6 M. Pumera, Chem. Commun., 2011, 47, 5671.
7 Y. Lu, X. Wang, D. F. Chen and G. Chen, Electrophoresis,
2011, 32, 1906.
8 Q. W. Chen, L. Y. Zhang and G. Chen, Anal. Chem., 2012, 84,
171.
A novel approach has been developed for the rapid preparation
of the graphene–CoMS hybrid at a low temperature of 80 ꢀC by
the borohydride-initiated reduction of GO sheets and cobalt(II)
chloride in a hydrazine-containing aqueous solution. CoMSs
have been successfully decorated on graphene nanosheets with
the diameter in the range from 500 to 800 nm. As precursors of
graphene, GO sheets act as two-dimensional growth templates
for CoMSs, resulting in the in situ deposition of CoMSs and
simultaneous reduction of GO nanosheets. The graphene–
CoMS hybrid displays ferromagnetic behavior at room-temper-
ature and should nd a wide range of applications. Moreover,
the magnetic hybrid has been successfully employed in the
enrichment and identication of low-abundance proteins and
peptides in combination with MALDI-TOF MS. The advantages
of the graphene–CoMS hybrid as an enrichment material
include excellent magnetic responsibility, large surface area,
high dispersibility, low expense of preparation, ease of opera-
tion, and satisfactory reproducibility, indicating great promise
for a wide range of applications.
9 W. D. Qu, L. Y. Zhang and G. Chen, Biosens. Bioelectron.,
2013, 42, 430.
10 K. S. Novoselov, V. I. Falko, L. Colombo, P. R. Gellert,
M. G. Schwab and K. Kim, Nature, 2012, 490, 192.
11 O. C. Compton and S. T. Nguyen, Small, 2010, 6, 711.
12 F. H. Li, H. F. Yang, C. S. Shan, Q. X. Zhang, D. X. Han,
A. Ivaska and L. Niu, J. Mater. Chem., 2009, 19, 4022.
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14 M. Giovanni, H. L. Poh, A. Ambrosi, G. Zhao, Z. Sofer,
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F. Sanek, B. Khezri, R. D. Webster and M. Pumera,
Nanoscale, 2012, 4, 5002.
15 H. T. Hu, X. B. Wang, F. M. Liu, J. C. Wang and C. H. Xu,
Synth. Met., 2011, 161, 404.
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Acknowledgements
17 H. B. Yang, G. H. Guai, C. X. Guo, Q. L. Song, S. P. Jiang,
Y. L. Wang, W. Zhang and C. M. Li, J. Phys. Chem. C, 2011,
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18 Q. Xie, Y. T. Qian, S. Zhang, S. Fu and W. Yu, Eur. J. Inorg.
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This work was nancially supported by the State Oceanic
Administration of China (201105007), NSFC (21375023 and
21075020) and Shanghai Science Committee (12441902900).
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