Detection of biomolecules via benign surface modification of graphene

Article abstract of DOI:10.1021/cm201577k

The modification of single layer graphene by assembly of a biotin moiety that is functionalized with a diazonium salt and the subsequent detection of Streptavidin (SA) upon exposure of graphene to compound V was highlighted. Graphene was prepared via CVD

Full text of DOI:10.1021/cm201577k

Communication
pubs.acs.org/cm
Detection of Biomolecules via Benign Surface Modification of
Graphene
Amal Kasry,*,†,‡ Ali A. Afzali,† Satoshi Oida,† Shu-Jen Han,† Bernhard Menges,§ and George S. Tulevski*,†
†IBM T. J. Watson Research Center, 1101 Kitchawan Rd, Yorktown Heights, New York 10598, United States ,
‡Egypt Nanotechnology Center (EGNC), Smart Village, Giza 12577 Egypt,
§Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
S
* Supporting Information
KEYWORDS: CVD graphene, surface modification, biosensor
raphene, since its isolation in 2004,1,2 has attracted a
∼600 Ohm/sq) upon exposure of graphene to compound V.
Gtremendous amount of interest because of its unique This mechanism is a critical advantage of this chemistry, as the
chemical and physical properties.3,4 Recent advances in large-
optical and electronic properties are retained after modification.
Compound IV was designed and synthesized (Scheme 1)
where the amine end group can be diazotized and assembled on
the graphene surface. Condensation of commercially available
N-Boc-4-aminobenzyl alcohol (II) with Biotin(I) in the
presence of dicylohexylcarbodiimide gave N-protected amino-
benzyl ester of Biotin(III) in 60% yield after purification as white
needles. Compound III was deprotected by stirring a solution of
III in dichloromethane containing 5 equivalents of trifluoroacetic
acid. Evaporation of the solvent followed by treatment with
sodium bicarbonate afforded the arylamine (IV) as a light yellow
powder, which was used in the next steps without further purifi-
cation. The synthetic route shown here is simple and
generalizable to virtually any desired sensing molecules.
Characterization of the synthesized compounds is shown in the
Supporting Information.
area growth and isolation of graphene has established it as a pro-
mising candidate for several potential applications,5−7 such as a
platform for biosensing.8 Adding functional groups in a con-
trolled manner is a critical first step in developing biosensors
that exploit graphene’s unique properties. There are several
reports on graphene functionalization.9−12 Graphene oxide and
exfoliated graphene are also widely used as a platform for
sensing.13−19 However, CVD graphene is a more desirable plat-
form because of the ease of fabrication, scalability, and its in-
herent low cost. In addition, this method allows for transfer of
graphene to any desired substrate.
The key results in this work include the modification of
single layer graphene (grown via CVD) by assembly of a biotin
moiety that is functionalized with a diazonium salt (compound
V, Scheme 1a) and the subsequent detection of Streptavidin
Graphene was prepared via CVD5 using a copper foil
annealed at 975 °C in 6 sccm forming gas (5% H2 in Ar) at a
pressure of 500 mTorr for 10 min, followed by flowing ethylene
for 10 min at the same temperature. The transfer method is
described elsewhere.20 The graphene was transferred to the
desired substrate and annealed at 600 °C in vacuum for 10 min.
The annealing aids in cleaning the graphene surface by
removing any traces of PMMA remaining from the transfer
procedure. The surface modification is achieved by the reaction
of V with the graphene surface. For the surface modification,
10 mg of compound IV was suspended in 10 mL of acetonitrile
mixed with 10 mg of nitrozonium tetrafluoroborate. The
graphene was then functionalized with compound V by ex-
posing a solution of V to the graphene, followed by washing
with copious amounts of ethanol and water.
Scheme 1. (a) Synthetic Scheme for Compound V; (b)
Cartoon Illustrating the Biotinylated Graphene and the
Selective Adsorption of Streptavidin Used in This Study
Compound V reacts with graphene via charge-transfer com-
plex formation were an electron is transferred from graphene to
compound V. The biotinylated graphene is then used to sense
streptavidin (SA) in solution. SA was bound to the surface
using a variety of optical techniques. Previous reports show that
diazonium salts react with graphene via charge-transfer com-
plexation, with very little covalent functionalization.11 The
compound merely shifts the Dirac point, decreasing the sheet
resistance as a result of charge transfer doping. The sheet resis-
tance decreases by a factor of 2 (from 1200 Ohm/sq to
Received: June 4, 2011
Revised: October 21, 2011
Published: October 28, 2011
© 2011 American Chemical Society
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by incubating the modified graphene for 15 min, at room
temperature, in solution of 1 μM SA in PBS.
Several techniques were used to characterize the system
described here. X-ray photoelectron spectroscopy (XPS) and
Raman spectroscopy measurements are shown in Figure 1. XPS
the most powerful techniques used for the detection of bio-
molecules on surfaces.24,25 Surface plasmons are collective
oscillations of the quasi free electron gas at the interface
between a metal and a dielectric. Absorption of molecules on
the metal surface leads to a change in the refractive index and,
consequently, a shift in the dispersion of the plasmon wave-
vector. This shift is used to extract information about the
thickness and/or the surface density of the absorbed mono-
layer. In this work, a single layer of graphene, of the size
1 cm × 1 cm, was transferred to a 2 nm/50 nm chromium/gold
film deposited on a glass coverslip. The sample was mounted
on the SPR set up in a Kretschman configuration as described
previously.26−28 Compound V was reacted with the surface, in
situ, using a 300 μL volume PDMS flow cell attached to the
substrate. The binding was monitored in real time by measuring
the change in the reflectivity of the gold substrate as a function
of time. The difference between the reflectivity signal before
binding and after washing with ethanol and water indicates the
degree of modification (Figure 2a). The binding of Streptavidin
Figure 1. (a) X-ray photoelectron spectra of the biotinylated graphene
showing an S2p doublet peak at 164.3 and 165.5 eV corresponding to
the sulfur atom in the compound V. (b) XPS spectra showing the N1s
peak at 401.3 eV corresponding to the Nitrogen atom in the Biotin.
(c) Raman spectra of the graphene before and after biotinylation
illustrating the increase in the D/G ratio.
Figure 2. Surface plasmon resonance spectra showing real time
binding of V with graphene. The difference between the baseline and
the signal after washing indicates the binding of compound V. (b) SPR
showing real-time binding of streptavidin to the biotinylated graphene.
Streptavidin remains on the surface even after washing with PBS
buffer.
was performed using a monochromatic source, Al Kα (hυ =
1486.6 e.v), and a photoelectron takeoff angle of 40°. The S2p
photoelectron spectrum, consisting of a doublet peak at 164.3
and 165.5 eV, is shown upon functionalization of graphene with
compound V. This is consistent with the presence of the sulfur
atom in the biotin moiety.21 A low intensity peak in the N1s
photoelectron spectrum that is consistent with previous reports
on the reaction of diazonium salts with graphene is also ob-
served (Figure 1b).22
Raman spectroscopy measurements were performed using a
Horiba ARAMIS Raman System with a HeNe laser source. The
Raman spectra of single-layer graphene has three key char-
acteristic peaks, the G peak, which his due to the vibrational
mode of sp2 bonded carbon; a 2D peak; and the D band, which
is induced by “defects” in the structure. Figure 1c contains
spectra of the graphene film before and after modification with V.
The key difference between the two spectra is the dramatic
increase in the ratio of the D/G after modification with V. The
D/G ratio increases dramatically after modification with com-
pound V. This is in agreement with studies where donor and
acceptor molecules are intercalated in few layer graphene
sheets.23 The charge transfer complex and resultant doping
leads to an increase in the D/G ratio. Although no deteri-
oration of the electrical properties is observed upon reaction
with compound V, there may be some conversion of sp2 to sp3
carbon, which contributes to the D band, but it is not
substantial enough to affect transport in these large area films as
evidenced by the decrease in sheet resistance following
modification.11
to the modified surface was also monitored by SPR. The bio-
tinylated graphene substrate was exposed to a 1 μM SA
solution. The real time measurements of SA binding are shown
in Figure 2b. Upon washing with PBS, there is no change in the
reflectivity signal due to the high affinity between Biotin and
the SA. Reflectivity scans were performed after each binding
step. Fitting of the reflectivity scans gave a SA surface density of
1.184 ng/mm2. Scanning electron microscopy (SEM) imaging
was used to determine the concentration dependence on SA
binding. This was done by modifying the graphene substrate
with the same concentration of compound V (40 mg/10 mL).
The biotinylated graphene was then exposed to differing
concentrations of Streptavidin-coated magnetic beads (Mitenyi
Biotecestimated concentration 1012 beads/mL) for 15 min.
Figure 3a is a plot of the number of beads as a function of their
concentration in solution, clearly indicating a concentration
dependence. The reaction begins to saturate at concentration
above 10%.
One of the most critical issues in developing viable biosen-
sors is preventing nonspecific binding of the biomolecules. Any
nonspecific binding can contribute to the detected signal and
give false positives. In our system, the graphene surface exhi-
bited no detectable nonspecific binding when the unmodified
graphene is exposed to streptavidin. This was shown by incu-
bating an unmodified and a biotinylated graphene layers in a
solution of the SA-modified magnetic beads. The comparison is
The surface modification was further examined via real time
measurements of compound V binding to graphene utilizing
surface plasmon resonance (SPR) spectroscopy. SPR is one of
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Chemistry of Materials
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ACKNOWLEDGMENTS
■
The authors thank Ageeth A. Bol from IBM research for the
help and the advice regarding the graphene growth. This work
was partially funded by the 2008 joint development agreement
between IBM Research and the Government of the Arab
Republic of Egypt through the Egypt Nanotechnology Center
(EGNC); http://www.egnc.gov.eg
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: akasry@us.ibm.com or amal.kasry@egnc.gov.eg
(A.K.); gstulevs@us.ibm.com (G.S.T.).
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Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
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