A graphene-based multifunctional affinity probe for selective capture and sequential identification of different biomarkers from biosamples

Article abstract of DOI:10.1039/c2cc35483g

A novel multifunctional graphene-based affinity probe has been explored for selective capture of two different types of peptides from the biosamples for sequential detection. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc35483g

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A graphene-based multifunctional affinity probe for selective capture and
sequential identification of different biomarkers from biosamplesw
Gong Cheng,ad Zhi-Gang Wang,ad Yan-Lin Liu,ad Ji-Lin Zhang,*a De-Hui Sunb and
Jia-Zuan Niac
Received 29th July 2012, Accepted 31st August 2012
DOI: 10.1039/c2cc35483g
A novel multifunctional graphene-based affinity probe has been
explored for selective capture of two different types of peptides
from the biosamples for sequential detection.
practical application. In addition, discrimination of phosphopep-
tides depends strongly on the tandem MS spectra or phosphatase
treatment currently which are indirect determination methods.7
Thus, multifunctional materials for selective capture, fast
isolation, and sequential determination of the low-abundance
peptides and the phosphopeptides from complex biosamples
are of great demand.
Many low-abundance endogenous peptides and phosphopeptides
in the body fluids or tissues are biomarkers with higher clinical
sensitivity and specificity, which could provide valuable information
for the detection of many diseases and elucidation of pathological
variations.1 Mass spectrometry (MS) is highly sensitive to trace
peptides in proteomics.2 However, it is still a great challenge to
detect the low-abundance peptides from complex biological samples
directly due to extremely low concentrations of these peptides and
interferences of high levels of small molecules such as salts,
surfactants and other contaminants in samples.3 In addition, detec-
tion of the phosphopeptides always suffers from the ion suppression
effect of nonphosphopeptides as well as the low ionization efficiency
of phosphopeptides due to the negatively charged sites.4 Thus, it is
essential to purify and separate these low-abundance peptides and
phosphopeptides prior to MS analysis.
Recently, graphene has been successfully utilized as an adsorbent
for MS detection of the low-mass molecules (e.g. amino acids,
polyamines, peptides, steroids, nucleosides and nucleotides)8 based
on its ultrahigh specific surface area, high loading capacity, unique
large delocalized p–p-electron system and hydrophobic inter-
action.9 Nevertheless, its poor hydrophilicity and difficult surface
functionalization often restrict its more extensive bioapplication,10
while the hydrophilic oxidizing graphite (GO) with its functional
groups located at both sides of the graphene sheet can be utilized as
the supporter for integration with many nanostructures rooted in
easy surface modification.11 It is easy to reduce the GO to graphene
(rGO) for the preparation of graphene-based multifunctional
materials. Compared with the carbon nanotubes used as
adsorbents for enrichment of the biomolecules (e.g. proteins
and long-chain peptides) by the interaction between carbon
atoms and biomolecules, the unique double-sided aromatic
chemical structure and high flexibility of graphene may produce
a more powerful capture of long carbon chains. These
characteristics also can be free from the hindrance of the
targeted analytes accessing the carbon nanotubes, and contribute
to more efficient adsorption.8 Most recently, our research results
indicate that the rare-earth affinity materials can selectively capture
and identify the phosphopeptides.12 Herein, a graphene-based
multifunctional composite material which combines the hydro-
phobic interaction of the graphene for enrichment of the low-
abundance peptides, the lanthanum phosphate affinity for detection
and identification of phosphopeptides and the Fe3O4 nanoparticles
for fast magnetic isolation has been designed and fabricated.
Such multifunctional composite material can realize selective
capture, fast magnetic separation and sequential determination
of the low-abundance peptides and the phosphopeptides from
the complex biosamples.
In recent years, some magnetic composites were explored to
enrich and separate low-abundance peptides or phosphopeptides
in object samples. For example, the C8-functionalized magnetic
particles were applied to enrich low-concentration peptides in
biological samples based on the hydrophobic interaction,5 while
affinity materials consisting of magnetic core and metal oxide shell
have been used for selective enrichment of the phosphopeptides
based on the selective affinity of the metal oxide for the phosphate
groups in the phosphopeptides.4,6 Although substantial progress
has been made in the low-abundance peptide and the phospho-
peptide enrichment, these composite materials cannot extract the
low-abundance peptides and the phosphopeptides synchronously.
Therefore, some useful and informative peptides/phosphopeptides
are lost during the enrichment step and no longer contribute to the
a State Key Laboratory of Rare Earth Resource Utilization,
Changchun Institute of Applied Chemistry, Chinese Academy of
Sciences, Changchun 130022, China. E-mail: zjl@ciac.jl.cn
b Changchun Institute Technology, Changchun 130012, China
c Key Laboratory of Marine Bioresources and Ecology, College of
Life Science, Shenzhen University, Shenzhen 518060, China
d Graduate University of the Chinese Academy of Sciences,
Beijing 100049, PR China
The synthesis strategy of the graphene-based multifunctional
affinity probe is shown in Scheme 1. The LaPO4–graphene oxide
composites (LaG) were first prepared by the electrostatic inter-
action between the negatively charged GO and the positively
w Electronic supplementary information (ESI) available: Experimental
details and further characterization data. See DOI: 10.1039/c2cc35483g
c
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Scheme 1 Schematic illustration of (a) the synthesis route to the
LaGM affinity probe and (b) the simultaneous capture and sequential
MS analysis of two types of peptides.
charged lanthanum ion and then the in situ growth of lanthanum
phosphate nanorods (Fig. S1, ESIw). After covalent grafting of
(3-mercaptopropyl)trimethoxysilane onto the GO (as verified by
FTIR in Fig. S2, ESIw), the Fe3O4 nanoparticles were readily
grafted to the GO via covalent Fe–S bonds. After reduction of the
GO, the multifunctional affinity probe (LaGM) with affinity,
hydrolytic catalysis, and strong magnetism as well as hydro-
phobicity rooted in the rGO (or graphene) was obtained.
Notably, the LaGM affinity probe not only can enrich the
low-abundance peptides from complex biosamples based on
the hydrophobicity from the unique structure of graphene used
as the supporter, but also can selectively capture and easily label
the phosphopeptides in virtue of affinity and hydrolytic catalysis of
the LaPO4 nanorods attached to the graphene. Furthermore, the
magnetic nanoparticles covalently grafted on the graphene facilitate
the separation and purification procedure. More importantly, the
two types of peptides can be selectively captured and then eluted
one by one for the sequential MS detection.
Fig. 1 (a) SEM image, (b–d) TEM images, (e, f) HRTEM images and
(g) EDS spectrum of the synthesized LaGM affinity probes.
in the XRD patterns of LaGM, indicating the existence of
magnetite. In addition, the appearance of the new absorption
peak at 565 cmꢀ1 (Fe–O–S) and the disappearance of the peak at
1735 cmꢀ1 (CQO) indicate covalent binding of the Fe3O4 and
reduction of the hydrophilic functional groups (e.g. –COOH) on
the GO. The magnetic properties of the LaGM and Fe3O4 were
examined via a superconducting quantum interface device
(SQUID) magnetometer (Fig. S4, ESIw), indicating a strong
superparamagnetism of LaGM at room temperature. Although
the saturation magnetization value of LaGM (25.1 emu gꢀ1) is
lower than that of Fe3O4 (77.6 emu gꢀ1), the LaGM can be
rapidly separated from the mixture within 1 min with the help of
a magnet (Fig. S4 inset, ESIw).
To examine the possibility of the LaGM probe for enrichment
of low-abundance peptides, the diluted BSA tryptic digest (10 nM)
was employed as a model sample. As shown in the MALDI-TOF
mass spectra (Fig. S5a, ESIw), before treatment only five peptides
(marked with ‘r’) with the low signal-to-noise (S/N) ratio can be
detected. However, after enrichment with the LaGM probe,
sixteen peptides with the increased MS S/N ratio and intensity
can be assigned to the peptides from the BSA tryptic digest
(sequence coverage of 27%, Table S1, ESIw), indicating the effec-
tiveness of the LaGM probe for enrichment of the low-abundance
peptides (Fig. S5b, ESIw). Furthermore, to inspect the selectivity of
the LaGM probe for peptides, human urine containing a high level
of salts and contaminants was used as a complex real biosample.
No peptides could be identified for direct detection of human urine
due to the interference of the high content salts and contaminants
(Fig. S6a, ESIw). However, five peptides with high S/N ratio and
intensity were observed after treatment with the LaGM probe
(Fig. S6b, ESIw), indicating that the LaGM probe can selectively
enrich the target peptides from complex biosamples.
SEM and TEM images (Fig. 1) show the triple hierarchical
structure of the multifunctional LaGM composites. Obviously, the
LaPO4 nanorods (ca. 300 nm in length and 10 nm in average
diameter) are evenly distributed on graphene, surrounding the
Fe3O4 nanoparticles (ca. 60 nm). Furthermore, no free Fe3O4
nanoparticles or LaPO4 nanorods are observed, indicating that the
Fe3O4 nanoparticles, the LaPO4 nanorods, and the graphene have
been integrated into an entity. According to high-resolution TEM
(HRTEM) images, the lattice fringe images of the LaPO4
nanorods (Fig. 1e) and the Fe3O4 nanoparticles (Fig. 1f) exhibit
the crystalline nature that can be indexed to the hexagonal
LaPO4 and cubic magnetite, respectively. The X-ray energy-
dispersive spectrum (EDS) (Fig. 1g) of the obtained LaGM
composites reveals the existence of C, O, La, P and Fe elements
(Cu arising from the sample carrier), further confirming the
triple hierarchical composites.
The formation of the LaGM triple hierarchical composites can
be further confirmed by powder X-ray diffraction (XRD) analysis
and Fourier-transform infrared (FTIR) spectroscopy. Compared
with the GO, new diffraction peaks in the XRD of LaG (Fig. S3a,
ESIw) that match well with those of the hexagonal LaPO4 (JCPDS
No. 04-0635) can be clearly observed, and the LaG shows a new
absorption peak in FTIR at around 1050 cmꢀ1 (O–P–O of LaPO4)
besides the characteristic absorption peaks at 1735 cmꢀ1 (CQO)
and 1621 cmꢀ1 (CQC) of graphene oxides (Fig. S3b, ESIw). After
attachment of the Fe3O4 and reduction of the GO, the diffraction
peaks of the magnetite phase (JCPDS No. 65-3107) can be observed
The LaGM probe can also be used for capture and labelling of
phosphopeptides based on the affinity and hydrolytic catalysis of
the LaPO4 nanorods, as shown in Fig. S7 (ESIw). To investigate
the phosphopeptide capturing and labeling efficiency, the b-casein
tryptic digest containing the phosphopeptides (Table S2, ESIw)
was first used as a model sample. Only one phosphopeptide with
weak intensity (marked with ‘*’) can be detected in the direct
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detected (Fig. 2a) in the first elution step, indicating the potential
of the LaGM probe for enriching low-abundance peptides from
real samples. More importantly, two labeled phosphopeptides are
successfully detected with a clean background in the second elution
step (Fig. 2b). The MALDI tandem mass spectrometry (MS/MS)
further confirms the effectiveness of the LaGM probe for capturing
and labeling the phosphopeptides (Fig. S12, ESIw). These results
demonstrate that the LaGM probe can be used for selective
capture, sequential MS detection and easy discrimination of
low-abundance peptides and phosphopeptides. For comparison,
two types of commercial affinity products (ZipTipC18 pipette tip
and TiO2 nanoparticles) were also used to capture target peptides
from diluted human serum under the same experimental conditions
(Fig. S10 and S11, ESIw). Obviously, the monofunctional
commercial products cannot be used for selective capture and
sequential determination of the corresponding biomolecules.
While the selectivity and the efficiency of commercial TiO2
nanoparticles for enrichment of phosphopeptides are inferior
to those of the LaGM probe.
Fig. 2 The MALDI-TOF mass spectra of the diluted human serum
solution: (a) the low-abundance peptides enriched by LaGM in the first
step, and (b) the labeled phosphopeptides captured by LaGM in the second
step. The number in the top right corner is the highest peak intensity.
analysis (Fig. S8a, ESIw) because of the nonphosphopeptide
suppression effect. However, after treatment with the LaGM probe,
three expected phosphopeptides with strong MS peak intensity are
detected (Fig. S8b, ESIw). Furthermore, these phosphopeptides can
be easily identified because a series of characteristic fragment ions of
mass loss of 80 Da (marked with ‘#’) are also observed. It should be
pointed out that the Fe3O4 nanoparticles also have corresponding
contribution to the capture of phosphopeptides. But then, their
contribution is far inferior to that of the LaPO4 nanorod based on
comparison of their MS intensities of phosphopeptides (Fig. S8c,
ESIw). To investigate the detection limit, the LaGM probe was
applied to capture phosphopeptides from the b-casein digest with
different concentrations. When the concentration is as low as
10ꢀ9 M, phosphopeptides can still be detected (Fig. S8d, ESIw).
A complex peptide mixture consisting of b-casein and BSA
(1 : 50 molar ratio) was further used to evaluate the selectivity
of the LaGM probe for capture of phosphopeptides. Appar-
ently, no phosphopeptides can be detected from the peptide
mixture due to the suppression effect and interference from the
abundant nonphosphopeptides (Fig. S9a, ESIw). However, after
selective capture using the affinity probe, three labeled phos-
phopeptides can be clearly identified in the mass spectrum and
free of the interference of nonphosphopeptide peaks (Fig. S9b,
ESIw). These results confirm that the LaGM probe can also be
used for selective capture of phosphopeptides.
In summary, a new graphene-based affinity probe consisting of
the graphene scaffold, affinity nanorods and magnetic nano-
particles has been synthesized. The multifunctional probe can be
used for selective capture, sequential determination and direct
identification of two types of peptides. This affinity probe has
better sensitivity and selectivity than the commercial ZipTipC18
pipette tip and TiO2 affinity product. Therefore, this work could
provide new insights for the design of the multifunctional
graphene-based affinity probes for extracting low-abundance
biomarkers from complex biosamples in biomedical application.
The work was supported by the Natural Science Founda-
tion of China (NSFC) (Grant No. 20871083 and 21171161).
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c
10242 Chem. Commun., 2012, 48, 10240–10242
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