A nanoscale graphene oxide-peptide biosensor for real-time specific biomarker detection on the cell surface

Article abstract of DOI:10.1039/c2cc31974h

A nanoscale RGD-pyrene-graphene oxide (GO) biosensor was prepared for real-time in situ detection of a cancer cell surface marker, integrin αvβ3. This nanoscale GO-based biosensor is simple, robust, sensitive and of high selectivity. It can also be adapted to other cancer cell surface marker evaluation systems. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc31974h

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A nanoscale graphene oxide–peptide biosensor for real-time specific
biomarker detection on the cell surfacew
Zhe Wang,abc Peng Huang,ab Ashwinkumar Bhirde,b Albert Jin,d Ying Ma,b Gang Niu,b
Nouri Neamati*c and Xiaoyuan Chen*b
Received 18th March 2012, Accepted 7th August 2012
DOI: 10.1039/c2cc31974h
A nanoscale RGD–pyrene–graphene oxide (GO) biosensor was
prepared for real-time in situ detection of a cancer cell surface
marker, integrin avb3. This nanoscale GO-based biosensor is
simple, robust, sensitive and of high selectivity. It can also be
adapted to other cancer cell surface marker evaluation systems.
So far, GO has been employed to detect various bio-
molecules. Noncovalent binding of GO with dye-labeled single
strand DNA (ssDNA) enables efficient detection of target
DNA in biological samples.10 GO can also be combined with
dye-tagged peptides to sense protease activities.13,14 In addition,
the modified GO has been used to sense ATP activity15,16 and
prostate specific antigen (PSA) detection.17 Although there are
many GO based biosensors available, the use of GO as a
sensitive biosensor for highly specific cancer cell surface
marker detection remains unexplored.
With the fast advancement of nanotechnology, many nano-
materials including quantum dots, carbon nanotubes,
magnetic nanoparticles, and gold nanoparticles or nanorods
etc., have been integrated with biomolecules for advanced
biomedical applications, such as drug/gene delivery,1 bio-
imaging2–4 and biosensing.5 Graphene, a 2D honeycomb
lattice consisting of sp2-hybridized carbons, has recently
drawn much attention due to its distinct electronic, mechanical,
optical and thermal properties from other carbon materials for
various applications.6 The oxidized counterpart of graphene,
graphene oxide (GO), provides a new type of aqueous dis-
persible biocompatible material for biological applications.
GO has been reported to be an effective drug delivery system
to carry poorly water soluble anticancer drugs for cancer
therapy.7,8 Due to the aromatic domain and multiple ionic
components of the basal plane, GO bears the unique capacity
to absorb biomolecules, such as nucleic acid9,10 or protein,11 to
behave as a sensitive biosensor. In addition, the sp2 aromatic
domain of GO results in the quenching effect of nascent
fluorescent dyes via fluorescence resonance energy transfer
(FRET) or dipole–dipole coupling effects.12 This property
facilitates GO to serve as a fluorescence quencher for fluores-
cence ‘‘on–off’’ biosensing.
As one of the widely used fluorescent dyes, pyrene possesses
large extinction coefficient, high quantum yield, and good
stability in aqueous media.18 Because of these attributes,
pyrene has been used as an optical reporter molecule for the
detection of infectious prion protein19 and DNA.20
Additionally, pyrene and its derivatives present noncovalent
interaction with molecules with a p-electron rich framework,
such as GO, carbon nanotubes, and fullerenes.21 Hence, the
combination of GO and pyrene provides an ideal platform for
biosensing. In this study, we report a GO-based biosensor to
efficiently detect cancer cell surface markers. As a proof-of-
principle, we employed cyclic RGD peptide and integrin avb3
as a ligand–receptor pair for GO based biosensing due to the
vital role of integrin in cancer cell adhesion, proliferation,
migration and metastasis.22 This GO based biosensor system is
initially at a quenching state due to the proximity of
RGD–pyrene to GO upon p-stacking interactions. However,
the competitive binding of an RGD receptor, integrin avb3,
to the RGD ligand disturbs the adsorption of RGD–pyrene
onto the GO surface, resulting in the recovery of pyrene
fluorescence. In addition to the detection of purified integrin
protein in buffer, which is a routinely used method under
many biosensor working conditions, we further demonstrated
the effectiveness for in situ detection of integrin overexpression
in live and fixed breast cancer cells.
a Center for Molecular Imaging and Translational Medicine,
School of Public Health, Xiamen University, Xiamen, China 361005
b Laboratory of Molecular Imaging and Nanomedicine,
National Institute of Biomedical Imaging and Bioengineering,
National Institutes of Health, Bethesda, MD 20892, USA.
E-mail: shawn.chen@nih.gov; Fax: +1 301-480-1613;
Tel: +1 301-451-4246
Scheme 1 illustrates the principle of RGD–pyrene–GO
biosensor for sensitive detection of integrin avb3 in solution
or on the cell surface membrane. A head-to-tail cyclic RGD
containing peptide, c(RGDyK), was used here due to its high
receptor binding affinity.2,23 The as-synthesized RGD–pyrene
conjugate (Fig. S1, ESIw) presents sol–gel character in aqueous
solution at concentration Z 3 mM. For cell surface marker
detection, we used a low concentration of this conjugate,
c Department of Pharmacology and Pharmaceutical Sciences,
University of Southern California, School of Pharmacy, Los Angeles,
CA 90089, USA. E-mail: neamati@usc.edu; Fax: +1 323-442-1390;
Tel: +1 323-442-2341
d Laboratory of Cellular Imaging and Macromolecular Biophysics,
National Institute of Biomedical Imaging and Bioengineering,
National Institutes of Health (NIH), Bethesda, MD 20892, USA
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc31974h
c
9768 Chem. Commun., 2012, 48, 9768–9770
This journal is The Royal Society of Chemistry 2012

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Fig. 1 Atomic force microscopy (AFM) images of nanoscale graphene
oxide before (a) and after (b) decorated with RGD–pyrene.
Scheme 1 Schematic illustration of RGD–pyrene interaction with
GO to quench the fluorescence of pyrene, and the fluorescence
recovery by introducing either integrin avb3 protein or integrin over-
expressing cancer cells. Size is not in scale.
2 mM, at which it is well-dispersed in solution. Upon addition
of RGD–pyrene into GO solution, the strong interaction of
pyrene with the basal plane of GO results in immediate
quenching of pyrene fluorescence due to the energy transfer
between GO and pyrene. Fig. S2a (ESIw) shows the UV/Vis
spectra of RGD–pyrene with GO. It can be seen that the GO
peak at 230 nm is slightly shifted to 232 nm in the RGD–
pyrene–GO complex, and the absorption peak of RGD–
pyrene at 348 nm is shifted to 351 nm. These slight red shifts
indicate the p–p interaction of pyrene with GO. To investigate
the dynamic interaction range of GO with the RGD–pyrene
conjugate, the absorption peak values at 232 nm were plotted
against the RGD–pyrene–GO complex concentrations
(Fig. S2b inset, ESIw). The extinction coefficient of the RGD–
pyrene–GO complex was estimated by Beer’s law from the
slope of linear square fit.20 It was found to be 0.0088 L mgÀ1 cmÀ1
with an R2 value of 0.9996. Fig. S2c (ESIw) shows the
fluorescence spectra of RGD–pyrene at different concentra-
tions with an excitation wavelength of 338 nm. The linear
relationship between the fluorescence intensity at the 400 nm
emission peak and RGD–pyrene concentration (Fig. S2c inset,
ESIw) confirms the good dispersion of the GO–RGD–pyrene
complex in aqueous solution. The atomic force microscopy
(AFM) images shown in Fig. 1 confirm the formation of the
GO–peptide complex with good dispersion, and maintenance
of the original nanoscale character of GO. The adsorption of
RGD–pyrene on GO resulted in 1–1.5 nm increase in thick-
ness, and marginal increase in size (Fig. 1b).
Fig. 2 Fluorescence spectra of RGD–pyrene in the presence of
various concentrations of GO (0, 0.1, 0.2, 0.4, 0.8, 1, 2, 5, 7,
10 mg mLÀ1). Inset presents the plot of fluorescence intensity at 380 nm
against the concentration of GO. lex: 338 nm.
fluorescence of pyrene significantly. In the subsequent studies,
2 mM RGD–pyrene and 2 mg mLÀ1 GO were used for integrin
biosensing.
In the presence of purified integrin avb3 in RGD binding
buffer (pH 7.4), the fluorescence intensity enhancement of
RGD–pyrene–GO was observed (Fig. S3, ESIw). This fluores-
cence recovery (FR) indicates that the competitive binding of
RGD–pyrene with integrin dissociates the p–p coupling and
hydrophobic interaction of GO with pyrene (Fig. S3a, ESIw),
and the recovered fluorescence intensity increased in accor-
dance with the ascending concentrations of integrin in buffer
(Fig. S3b, ESIw). To study the binding specificity of this
RGD–pyrene probe to integrin avb3, we investigated the
fluorescence recovery of RGD–pyrene in various biological
samples. As shown in Fig. 3, the probe exhibited little FR to
Next, we investigated the quenching effect of GO on the
fluorescence of the RGD–pyrene conjugate. Fig. 2 shows the
concentration dependent fluorescence quenching effect of GO
on RGD–pyrene in aqueous solution. An exponential reduction
of RGD–pyrene was observed with increasing concentration
of GO. The quenching efficiency of GO is calculated by using
the formula: QE = (1 À a) Â 100%, where a is the ratio of
fluorescence quenched to the original fluorescence of the
RGD–pyrene conjugate. Hence, the QE of GO to the RGD–
pyrene conjugate (2 mM) is calculated to be 96.2 Æ 0.13%.
Higher concentrations of GO did not further quench the
Fig. 3 Specificity of integrin in recovering the fluorescence of the
RGD–pyrene–GO complex.
c
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Chem. Commun., 2012, 48, 9768–9770 9769

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(Fig.S6 and S8, ESIw). In contrast, fluorescent RGD–pyrene
without GO showed different cellular uptake behavior in
which the GO free RGD–pyrene probe diffused into cyto-
plasm upon integrin binding (Fig. 4c,d and Fig. S6–S8, ESIw)
after 2 h incubation. This is partially due to the hydrophobi-
city of pyrene. In contrast, the RGD–pyrene–GO probe bound
to the cell surface membrane (Fig. 4a,b, white arrow,
Fig. S6–S8, ESIw). Flow cytometry results (Fig. S5, ESIw)
confirmed the above phenomena. This specific cell receptor
binding study provides the initial evidence that the RGD–
pyrene–GO complex is a suitable probe for real-time cancer
cell integrin expression detection.
In conclusion, we have designed a simple, robust graphene
oxide (GO) based fluorescent biosensor for efficient, selective
and real-time cancer cell surface marker integrin avb3 detec-
tion. We envision that this RGD–pyrene–GO biosensing
system can be extended to evaluate other cell surface markers
for cell surface marker imaging or ligand screening.
Fig. 4 Real-time in situ detection of breast cancer cell surface integrin
expression by the RGD–pyrene–GO probe: (a) probe fluorescence
recovery by live MDA-MB-435 cancer cells which overexpress integrin
avb3 on the cell surface. The recovered fluorescence is mainly detected
on the cell membrane as indicated by white arrows; (b) probe
fluorescence recovery by MDA-MB-435 cancer cells followed by 4%
formalin fixing; (c) equivalent concentration of free RGD–pyrene
incubated with live MDA-MB-435 demonstrates significant endocytosis
as indicated by white arrows; (d) equivalent concentration of RGD–
pyrene incubated with MDA-MB-435 followed by 4% formalin fixing.
Notes and references
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9770 Chem. Commun., 2012, 48, 9768–9770
This journal is The Royal Society of Chemistry 2012