Electrochimica Acta 290 (2018) 356e363

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Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta

A graphene aptasensor for biomarker detection in human serum

Xuejun Wang a, b, Yibo Zhu b, Timothy R. Olsen b, Na Sun c, Wenjun Zhang a, d, Renjun Pei c,
Qiao Lin b, *
a
Department of Mechanical Engineering, East China University of Science and Technology, Shanghai, 200237, China
b
Department of Mechanical Engineering, Columbia University, New York, NY 10027, USA
c
Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, Jiangsu, 215123, China
d
Department of Mechanical Engineering, University of Saskatchewan, Saskatoon, S7N 5A9, Canada

a r t i c l e i n f o a b s t r a c t

Article history: This paper presents an affinity graphene nanosensor for detection of biomarkers in undiluted and non-
Received 24 April 2018 desalted human serum. The affinity nanosensor is a field-effect transistor in which graphene serves as
Received in revised form the conducting channel. The graphene surface is sequentially functionalized with a nanolayer of the
10 July 2018
polymer polyethylene glycol (PEG) and a biomarker-specific aptamer. The aptamer is able to specifically
Accepted 29 August 2018
bind with and capture unlabeled biomarkers in serum. A captured biomarker induces a change in the
Available online 13 September 2018
electric conductivity of the graphene, which is measured in a buffer of optimally chosen ionic strength to
determine the biomarker concentration. The PEG layer effectively rejects nonspecific adsorption of
Keywords:
Affinity sensing
background molecules in serum while still allowing the aptamer to be readily accessible to serum-borne
Biomarker detection biomarkers and increases the effective Debye screening length on the graphene surface. Thus, the
Graphene aptamer-biomarker binding sensitively changes the graphene conductivity, thereby achieving specific
Nonspecific binding and label-free detection of biomarkers with high sensitivity and without the need to dilute or desalt the
Human serum serum. Experimental results demonstrate that the graphene nanosensor is capable of specifically
capturing human immunoglobulin E (IgE), used as a representative biomarker, in human serum in the
concentration range of 50 pMe250 nM, with a resolution of 14.5 pM and a limit of detection of 47 pM.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction specific detection of target molecules of interest can be achieved

Early detection of disease biomarkers is highly sought after in
clinical diagnostics. Biosensors that utilize nanomaterials, such as
conducting polymer nanowires [1], semiconductor nanowires [2],
carbon nanotubes [3], and graphene [4], offer attractive advantages
for biomarker detection. In particular, graphene, a two-dimensional
honeycomb lattice of carbon with a sp2 structure, offers unique
physical and chemical properties such as high intrinsic carrier
mobility, large surface-area-to-volume ratio, low intrinsic noise,
and good biocompatibility [5e7]. Biosensors using graphene have
been demonstrated on optical [8,9], optomechanical [10], and
electrochemical [11,12] platforms. Of particular interest are field
effect transistor (FET) nanobiosensors using graphene as the con-
ducting channel [4,13], allowing target detection with high sensi-
tivity. When combined with an appropriate recognition molecule,
* Corresponding author.
E-mail addresses: wxjecust@gmail.com (X. Wang), qlin@columbia.edu (Q. Lin).
[14]. One important advance in graphene FET nanosensors involves
using aptamers, single-strand oligonucleotides with high affinity
and specificity to a biomarker, as the recognition element [4].
Aptamers, compared to other receptor molecules such as anti-
bodies, have attractive advantages such as synthetic availability,
small size, batch-to-batch uniformity, stability and long shelf life,
for effective surface-based detection [15]. As a result, aptamer-
based graphene FET devices have been used for the detection of a
variety of biomarkers such as DNA [16], peptides [17], and proteins
[4].
Previously reported aptameric FET sensors, however, have been
mostly limited to the detection of biomarkers in conditioned
buffers. In complex physiological media such as serum, plasma or
whole blood, abundant background molecules tend to adsorb
nonspecifically to the hydrophobic graphene surface [18] and thus
interfere with biomarker detection. Such interferences may cause
difficulties in accurately measuring biomarkers. For example, a
graphene oxide (a graphene derivative) FET nanosensor [19] was
employed to measure prostate cancer biomarker in serum, and

https://doi.org/10.1016/j.electacta.2018.08.062
0013-4686/© 2018 Elsevier Ltd. All rights reserved.
 X. Wang et al. / Electrochimica Acta 290 (2018) 356e363
although it was able to demonstrate the successful quantification, it
also experienced appreciable adsorption of serum proteins because
of the interaction between serum proteins and the reduced gra-
phene oxide [20]. Moreover, either dilution or desalting of serum,
which may reduce the measurement sensitivity of low concentra-
tion biomarkers or increase the complexity of the protocol while
potentially compromising the viability of the analyte, was neces-
sary. Further challenging these sensors is the short Debye screening
length (<1 nm), the characteristic thickness of the electrical double
layer, resulted from serum's high ionic strengths which can cause
the binding events to occur outside the electrical double layer and
drastically reduce the biomarker detection sensitivity [21]. Thus,
despite its importance, the detection of biomarkers directly in
serum or in other forms of physiological media using graphene FET-
based nanosensors has yet to be demonstrated.
This paper presents an affinity graphene nanosensor for the
detection of biomarkers in undiluted and non-desalted human
serum. The affinity nanosensor employs an FET-based design in
which graphene serves as the conducting channel. The graphene
surface is sequentially functionalized with a nanolayer of the
polymer polyethylene glycol (PEG) and a biomarker-specific
aptamer. The aptamer binds with and captures biomarkers, which
are not tagged with any labeling groups, in serum. The captured
biomarker induces a change in the electric conductivity of the
graphene, which is measured via a solution-gated FET formed in a
buffer of optimally chosen ionic strength to determine the
biomarker concentration. In this approach, the sequential attach-
ment of the aptamer and PEG to the graphene surface allows the
aptamer to be readily accessible to serum-borne biomarker mole-
cules, while the PEG effectively prevents nonspecific adsorption of
background molecules in serum [22] and protects graphene from
the interferences of such molecules. In addition, the small size of
the aptamer facilitates the captured biomarker molecules to stay in
the close proximity of the graphene, and the immobilized PEG in-
creases the effective Debye screening length [23e25]. Conse-
quently, the biomarker is readily recognized in serum by the
aptamer, and their binding sensitively changes the graphene con-
ductivity, thereby achieving the specific and label-free detection of
the biomarker with high sensitivity.
The capability of the nanosensor is demonstrated by detection
of the protein immunoglobulin E (IgE), chosen as a representative
biomarker, in undiluted and non-desalted human serum. Repro-
ducible measurements of IgE at concentrations ranging from 50 pM
to 250 nM are made with an estimated resolution of 15.6 pM and
limit of detection of 47 pM. These clinically relevant measurements
demonstrate the potential of our approach for biomarker detection
in human serum and other forms of physiological media.
2. Experimental method
2.1. Principle and design
The graphene nanosensor is configured as a solution-gated
graphene FET, where the graphene conducting channel is sequen-
tially functionalized first with a PEG nanolayer and then with
aptamer molecules. PEG is a hydrophilic, electrically neutral poly-
mer that can inhibit nonspecific adsorption of background mole-
cules in physiological samples that are typically hydrophobic and
charged [26]. The immobilized PEG polymer on graphene surface
forms a porous and biomolecule permeable layer on the surface of
FET while also changing the dielectric properties in aqueous solu-
tions, which increases the effective Debye screening length [24].
The aptamer is attached to the PEG that is in turn tethered to the
graphene surface, thereby leaving the aptamer exposed to
biomarker molecules in the sample with the PEG thickness tunable
357
for optimization of the adsorption rejection and detection sensi-
tivity. Thus, when exposed to a serum sample, the functionalized
PEG nanolayer reduces the nonspecific adsorption of background
molecules (Fig. 1) while the attached aptamer specifically captures
the biomarker in serum. As it binds to the biomarker, the aptamer
experiences a conformational change, bringing the biomarker into
the proximity of the graphene surface. In this configuration, an
electrical double layer is formed at the interface of the graphene
and the electrolyte serves as the gate dielectric layer. The charged
target molecule induces a change in the electrical conductance of
the graphene through the electrical double layer, thereby produc-
ing a detectable signal. The graphene channel is formed between
drain and source electrodes on the SiO2 substrate as shown in
Fig. 2(a). Target molecules are in solution (contained within a
polydimethylsiloxane open well located above the sensor) and
allowed to interact with the aptamer linked to the nanolayer of PEG
that is in-turn attached to the graphene surface. An Ag/AgCl wire
inserted into the solution to forms the gate electrode. Given the
experimental scheme of graphene FET nanosensors where the
sample solution in the graphene sensing region is exposed to the
ambient environment, the exposure of serum to air leads to the
reduction of CO2 from the serum, changing the pH of serum over
the time. The pH variation, to which the graphene is highly sensi-
tive, brings inaccuracy to the biomarker detection. In this work, the
nanosensor operates scheme that decouples the capture and
detection of the biomarker. That is, the biomarker is sequentially
first captured by the aptamer in serum, which in general involves
diversely varying high ionic strength and pH ranges, and then
detected in conditioned buffer with well-controlled ionic strengths
and pH to enable optimized FET measurements [27].
2.2. Device fabrication and surface functionalization
The device was fabricated using micro and nanofabrication
techniques (see Fig. S1 in Supplementary Information). Graphene
synthesized via chemical vapor deposition (CVD) was transferred
onto the target substrate using a poly methyl methacrylate carrier
layer (PMMA), following a previously reported protocol [16], which
pre-fabricated source and drain electrodes (Fig. 2(b)). Then the
PMMA carrier layer was removed by acetone. The graphene then
was patterned to define the sensing region (See Fig. S2 in Supple-
mentary Information). Finally, a polydimethylsiloxane (PDMS)
open well (~10 mL in volume) was stamped above the graphene
sensing region to hold the target solution. Before the surface
functionalization, the graphene was verified to be of a monolayer
via Raman spectroscopy (Renishaw inVia confocal Raman micro-
scope). The graphene channel was then biochemically functional-
ized (see details in Supplementary Information) to achieve the
target molecule sensing in serum (Fig. 2(c)). PEG and aptamer were
serially functionalized on graphene surface via a 1-pyrenebutanoic
acid succinimidyl ester (PASE) linker, which was already non-
covalently coupled to graphene surface by p-p interactions.
2.3. Testing procedure
During operation, the modified graphene conducting channel
was exposed to IgE-spiked serum solutions at different concen-
trations for 10 min respectively. After this incubation period, the
sensor was gently washed with buffer and the open chamber was
filled with 0.01
fresh PBS buffer. A bias drain-source voltage Vds
applied between the drain and source electrodes generated a drain-
source current Ids through the graphene channel, and the Ids was
controlled by the applied gate voltage Vg. Transfer characteristics
(i.e., the functional dependence of Ids on Vg) of graphene was
measured by sweeping the Vg in a range of interest, and the Dirac
358 X. Wang et al. / Electrochimica Acta 290 (2018) 356e363

Fig. 1. Principle of nanosensing in serum. (a) PEG and aptamer immobilized graphene. (b) Serum introduction. (c) IgE capture and purification on the graphene surface, impurities
repelled by grafted PEG.

Fig. 2. Design, fabrication and functionalization of the graphene nanosenor. (a) The device is configured as a liquid-gated graphene FET. (b) Micrograph of a fabricated device. The
patterned graphene conducting channel connects the drain and source electrodes (Inset: photograph of the sensor chip). (c) Surface Functionalization of graphene. Pyrene-
terminated PASE linker molecules are coupled to graphene via p-p interactions. PEG and aptamer molecules are sequentially attached to the PASE by forming amide groups.

point VDirac, the voltage at which the Ids reached its minimum, were
measured. In the FET-based electrical measurement, both the drain
and gate voltage were supplied by sourcemeters (Keithley 2400,
Tektronix), and the drain-source current was simultaneously
measured. The two sourcemeters were automatically controlled
through a PC-based LabVIEW program.
2.4. Materials
Graphene was synthesized via chemical vapor deposition (CVD).
Polydimethylsiloxane (PDMS, Sylgard-184) was purchased from
Dow Corning (Midland, MI) for PDMS well. 1-pyrenebutanoic acid
succinimidyl ester (PASE) linker and phosphate buffered saline
 X. Wang et al. / Electrochimica Acta 290 (2018) 356e363
(PBS) were purchased from Thermo Fisher Scientific (Waltham,
MA). Amine-PEG-carboxylic acid, 50 -amino group modified IgE DNA
aptamer D17.4 [28] (selective sequence 50 -NH2 GCG CGG GGC ACG
TTT ATC CGT CCC TCC TAG TGG CGT GCC CCG CGC-30 ) and its
complementary DNA oligonucleotide (selective sequence 50 -FAM
GCG CGG GGC ACG CCA CTA GGA GGG ACG GAT AAA-30 ) were
synthesized and purified by Integrated DNA Technologies (Coral-
ville, IA). Target molecule, Immunoglobulin E (IgE) and control
molecule, Immunoglobulin G (IgG) were purchased from Athens
Research and Technology (Athens, GA). 1-ethyl-3-(3-
dimethylaminepropyl carbodiimide hydrochloride (EDCHCl) and
N-hydroxysulfosuccinimide (NHS), Dimethylformamide, ethanol-
amine and PBS as well as other chemicals used in functionalization
and experiments were obtained from Sigma-Aldrich (St. Louis, MO).
Human serum was obtained from Dr. Worgall in the Department of
Pathology and Cell Biology Medical, Columbia University.
3. Results and discussion
3.1. Surface characterization
Prior to functionalizing the graphene surface, we characterized
the properties of the graphene specimen used to fabricate the
nanosensor with a Raman microscope under the 532-nm laser
excitation. In its Raman spectra (Fig. 3), the G band at 1587 cm1
and 2D band at 2679 cm1 are observed. These two bands were
signature peaks of all graphitic sp2 materials. The intensity ratio of
2D band to the G band I2D =IG is 3.75, which was the further evi-
dence of monolayer graphene [29]. The PASE functionalization
splits the G band (1587 and 1626 cm1), confirming the coupling of
the graphene and pyrene groups on PASE [30]. The PASE immobi-
lization shifted the 2D band to a higher wavenumber (from 2679 to
2684 cm1), which was typical for PASE chemical doping [13]. In
addition, the prominent D band at 1342 cm1 compared with
pristine graphene indicated the enhanced disorder of the graphene
structure after the coupling of PASE molecules.
We next investigated the serial functionalization of the gra-
phene surface with the PEG and the IgE aptamer. Fig. 4 depicted
atomic force microscopy (AFM) images of single-layer graphene
Fig. 3. Raman spectra of graphene before and aptamer PASE functionalization.
Signature peaks of p-p coupling between PASE and graphene are observed after PASE
treatment.
359
and the aptamer-PEG-modified graphene. The height of graphene
sheet after surface functionalization had a significant increase from
0.4 nm of pristine graphene in Fig. 4(a) to 6.1 nm of modified gra-
phene in Fig. 4(b). Such a morphology change illustrated a suc-
cessful functionalization of PEG and aptamer molecules.
Furthermore, graphene surfaces functionalized with PEG and the
aptamer, and graphene functionalized with only PEG were incu-
bated with a solution containing fluorescently modified single-
stranded DNA complementary to the aptamer and washed with
buffer. The background-subtracted fluorescent intensities of the
graphene surfaces were measured. The graphene surface modified
with both PEG and the aptamer exhibited strong fluorescence in-
tensity after incubation with the single-stranded DNA (~28.8 a.u.),
while the graphene surface functionalized with only PEG showed
negligible fluorescent intensity (~0.9 a.u.) (Fig. 5). Since the
fluorescence-dyed single-stranded DNA only hybridized to the
complementary aptamer, the fluorescence signal presented in the
PEG and aptamer functionalized graphene confirmed the successful
functionalization of PEG and aptamer on graphene.
3.2. Rejection of nonspecific adsorption
The ability of PEG to reject nonspecific adsorption of background
molecules in serum to the nanosensor surface was characterized by
measuring the transfer characteristics of bare or PEG-coated gra-
phene surface in PBS buffer before and after incubating the surface
in serum (Fig. 6). Incubation of bare graphene in serum reduced the
Dirac point VDirac by approximately 50 mV (Fig. 6(a)), which was
believed to reflect the direct doping caused by the accumulated
background molecules adsorbed to graphene surface. In contrast,
the PEG modified graphene did not exhibit any appreciable shift of
VDirac after serum incubation (Fig. 6(b)), suggesting a significant
reduction of nonspecific adsorption. This demonstrates the effec-
tiveness of the PEG modification to repel non-target molecules. In
general, despite the same layout of devices, graphene sheet used in
each device had different electronic properties (e.g., carrier density
and carrier mobility), leading to variations in the shape and
magnitude of the transport characteristic curves (Fig. 6). Fortu-
nately, such device-to-device variations did not significantly impact
the VDirac, which was hence used as the primary sensor output.
3.3. IgE detection in serum
The graphene nanosenor was then used to quantitatively mea-
sure the concentration of IgE spiked in human serum. When the
device was exposed to serum solutions with increasing IgE con-
centrations from 50 pM to 250 nM as shown in Fig. 7(a) followed by
gentle buffer rinsing and measuring in buffer, the Dirac point VDirac
monotonically decreased towards the negative gate voltage from
0.298 to 0.268 V, suggesting that the binding of the aptamer to IgE
introduced n-type doping to the graphene. The measurements
were carried out in 0.01
PBS buffer, for which the Debye length
was estimated to be approximately 7.3 nm [31]. In our experiments,
the Debye length would be even larger than this value due to the
presence of the PEG layer [24]. On the other hand, AFM measure-
ment showed that the aptamer-IgE complex was roughly 5.7 nm
above the graphene surface (Fig. 4). Therefore, the binding of IgE
with the aptamer should have at least partially taken place within
the electrical double layer as evidenced by the sensor's sensitive
response.
The physiochemical process involved in the IgE-aptamer bind-
ing is complex; thus the doping effects may be a result of two
molecular interactions involving graphene. Theses two possible
mechanisms, which combine to contribute to the observed sensor
response, are discussed below. Further studies are needed in the
360 X. Wang et al. / Electrochimica Acta 290 (2018) 356e363

Fig. 4. AFM images of graphene surface before and after PEG and aptamer functionalization. (a) AFM image of pristine graphene, the 0.4 nm height indicates single-layer graphene.
(b) AFM image of aptamer-PEG-modified graphene, the 6.1- nm thickness suggests the successful PEG and aptamer functionalization.

Fig. 5. Characterization of surface functionalization. Both PEG-modified graphene and

PEG-aptamer-modified surfaces is exposed to a fluorescence-labeled complementary
DNA sequence to IgE-specific aptamer. Complementary DNA sequence hybridizes to
the aptamer-PEG-graphene surface, but not the PEG-graphene surface.

future to understand and elucidate these mechanisms. First,

Detection of large biomarkers, such as proteins, using an
electrolyte-gated graphene FET is typically based on the electro-
static gating effect generated by the highly charged biomarkers on
graphene. Under these circumstances, the charges induced in gra-
phene are opposite to and hence in balance with the charges car-
ried by target molecules. In our experiments, as IgE is reported to
have overall positive charge at physiological pH [4], the binding of
IgE proteins to the aptamer induces charges in graphene that were
opposite to the charges carried by the target molecules, as a result
of electrostatic gating effect. As such, n-type doping of graphene
occurs. Second, upon the capture of IgE, the aptamer experiences a
large change in conformational structure, becoming a stable
compact structure. Conformational change of the aptamer brings
electron-rich aromatic nucleotides to a close proximity of the gra-
phene surface, resulting in direct interactions of nucleotide with
Fig. 6. Measured transfer characteristics of bare graphene and PEG-modified graphene
the graphene and making direct electron transfer more likely from
before and after serum incubation. (a) Bare graphene. Significant shift of VDirac in-
the backbone of aptamer into graphene through pep interactions dicates nonspecific adsorption of background molecules in serum onto graphene
between them [6,32]. Such a claim can be corroborated by the surface. (b) PEG-modified graphene. No appreciable shift occurs, demonstrating the
physical process observed in the DNA immobilization on carbon rejection of nonspecific adsorption by PEG nanolayer.
 X. Wang et al. / Electrochimica Acta 290 (2018) 356e363
Fig. 7. Quantitative measurement of IgE in human serum. (a) Measured transfer
characteristics of the graphene nanosensor exposed to human serum spiked with IgE
(concentration range: 50 pM e250 nM). (b) Shift of the Dirac point (DVDirac) as a
function of the IgE concentration (CIgE) (three devices tested). The solid curve shows a
fit to the Hill-Langmuir equation, yielding an equilibrium dissociation constant of
KD ¼ 10.7 nM. (c) Linear fit of the drain-source current change DIds for the linear range
from 50 pM to 25 nM at fixed Vg ¼ 0.2 V.
361
nanotube [33] and graphene oxide sheet [34], which possibly
serves as the underlying mechanism for the sensor response. As
such, despite the heavily negatively charged DNA aptamer, the
screening of DNA charge is believed to be negligible compared with
the direct electron transfer. The specificity of the graphene nano-
sensor was confirmed by examining its response in serum spiked
with immunoglobulin G (IgG) and immunoglobulin A (IgA), which
were used as control molecules. IgG and IgA are similar to IgE in
structure and thus can serve as appropriate control proteins for the
aptamer binding specificity. The transport characteristic curves
showed negligible shift for both IgG and IgA molecules (See Fig. S4
in Supplementary Information), suggesting the specificity of the
graphene nanosensor to IgE molecules. This could be attributed to
the high specificity of the aptamer to IgE and the effective resis-
tance of PEG to nonspecific adsorption. The specificity of the
nanosensor was also supported by previous studies of the low
cross-reactivity (<5%) of the IgE-targeting D17.4 aptamer, which
was used to functionalize the nanosensor, to proteins that exist at
high concentrations in biological samples such as human serum
albumin and bovine serum albumin [35].
We performed the IgE capture in serum and FET measurement
in diluted PBS buffer. This use of different media required the
replacement of serum with diluted PBS buffer during the sensor
operation. Such an operation scheme could initiate the dissociation
of IgE molecules from the aptamer. Thus, we conducted a time-
resolved measurement of VDirac after the replacement by consec-
utively sweeping the gate voltage for 10 min and measuring the
drain current of the graphene conducting channel continuously
over time (see Fig. S5 in Supplementary Information). The experi-
mental results showed, over a period of 1 min, an overall shift in the
Dirac point that was 0.83% of that induced by IgE binding, As the
measurement of the graphene transport characteristic curve of was
completed well within 1 min, we concluded that the effect of IgE
dissociation from the aptamer during medium replacement was
negligible for purposes of IgE quantification. Such a negligible
dissociation we believe can be attributed to that, the electrical
measurement was performed in buffer at static flow conditions that
reduced dissociation since the process was limited by the time for
the dissociated IgE molecules to diffuse back to the bulk solution. It
has been studied that electrostatic interactions between salts ions
and negative-charged phosphate backbones of DNA facilitate the
dissociation of aptamer-IgE complex [36,37]. Hence, the diluted PBS
buffer with low salt-ionic strength used for measurements could
further lower the dissociation rate of the aptamer-IgE complex.
It can be observed in Fig. 7(a) that transconductance (dIds/dVg)
of both hole and electron conduction branches remain constant, at
~20.2 mS and ~16.3 mS respectively. Given that the ionic strength of
the buffer did not change, the electrical double layer capacitance
was also deemed unchanged by the IgE-aptamer binding [38]. We
also investigated the change of source-drain current Ids at fixed gate
voltage Vg. It was observed that the Ids either decreased or increased
monotonically with increasing IgE concentration depending on
whether the Vg was lower or higher than the VDirac, such reversed
electrical transport in different conduction band is due to the
ambipolar transport of graphene that can be described by the
following equation [39]:


Ids ¼ mðW=LÞCVds Vg  VDirac (1)
where C is the top-gate capacitance per unit area, m is the carrier
mobility, W and L are respectively the width and length of the
graphene channel, and VDirac is voltage indicating Dirac point (Ids
reaches the minimum). Experiments with three different devices
were carried out to characterize the sensing performance of our
graphene nanosensor. The Dirac point shift (DVDirac) as a function of
362 X. Wang et al. / Electrochimica Acta 290 (2018) 356e363
IgE concentration (CIgE) is presented in Fig. 7(b). The error bars
represent standard deviations of DVDirac, which mainly resulted
from variations in the electrical and physicochemical properties of
the individual devices tested. It can be seen that DVDirac sharply
increased for CIgE ranging from 50 pM to 25 nM and then gradually
reached the equilibrium state for CIgE above 160 nM. We fitted the
experimental results to the following Hill-Langmuir equation that
describes the equilibrium ligand-receptor binding [4].
DVDirac;max CIgE
DVDirac ¼ (2)
KD þ CIgE
where DVDirac,max and CIgE are the saturated shift of VDirac and IgE
concentration respectively. As the fitted Hill-Langmuir curve lied in
the error bars, our graphene nanosensors show good repeatability
of the IgE detection in human serum. The fitted curve yielded an
equilibrium dissociation constant KD of 10.7 nM, in good agreement
with previously reported work [40]. We further studied the
decrease of Ids with increased IgE concentration at an appropriate
gate voltage Vg ¼ 0.2 V (Fig. 7(c)). By performing a linear fit to the
measurement data in the linear range (50 pM- 20 nM), the sensi-
tivity of the nanosensor, defined as DIds/DCIgE, or the shift of Ids
induced by unit IgE concentration, was determined to be 14.5 nA/
nM. This sensitivity, along with the estimated noise level of
0.227 nA for the measurement system, was used to determine the
limit of detection (LOD), the lowest IgE concentration that could be
reliably detected: LOD ¼ (S/N x Noise)/Sensitivity z 47 pM, where a
signal-to-noise ratio of S/N ¼ 3 was used. The estimated LOD of 47
pM is clinically relevant [41], and it is comparable to previous re-
ported detection limit for IgE in buffer by carbon nanotube and
quartz crystal microbalance sensors [42,43]. Also, when compared
with traditional microfabricated semiconductor FET biosensors
that have been reported to offer detection limits on the order of
micromolars for detection of proteins in buffer [44], this LOD, in
more complex samples (serum), of the graphene FET nanosensor
represents a drastic improvement.
4. Conclusions
We have presented a graphene-based affinity nanosensor for
biomarker detection in human serum, using IgE as a representative
biomarker. The nanosensor employed an FET-based design in
which graphene in which graphene was sequentially functionalized
with PEG and aptamer. The sequential aptamer-PEG functionali-
zation scheme enables the aptamer to be readily accessible to the
biomarker in serum, while the PEG reduces or rejects the
nonspecific adsorption of background molecules. This graphene
nanosensor operated in a scheme where the capture and detection
of the biomarker were decoupled, with the biomarker captured
from serum and FET measurements of the resulting biomarker-
aptamer complex in diluted buffer to provide reasonable Debye
length, which was further increased by the immobilized PEG
nanolayer, allowing the aptamer-biomarker binding to induce a
significantly measurable change in the graphene's electric con-
ductivity. As we recognize the complexity of the sensor response,
we believe that two possible mechanisms combines to contribute
to the observed nanosensor response including the electrostatic
gating effect from captured IgE molecule and direct electron
transfer from aptamer to graphene. Experimental results showed
that the scheme of sequential functionalization of the PEG and
aptamer effectively rejected the nonspecific adsorption of back-
ground molecules in serum to the graphene surface. The graphene
nanosensor was tested in human serum spiked with IgE at con-
centrations ranging from 50 pM to 250 nM, yielding a limit of
detection of 47 pM. These characteristics are clinically relevant and
demonstrate the potential of the graphene nanosensor for specific,
quantitative and label-free detection of biomarkers in physiological
fluids.
Acknowledgements
This work was supported by the National Science Foundation,
United States (Grant No. ECCS-1509760) and National Institutes of
Health (Grant No. 1R33CA196470-01A1). X. Wang gratefully ac-
knowledges a National Scholarship (Award No. 201406740003)
from the China Scholarship Council. The authors would also like to
thank Dr. Tilla S. Worgall at the Department of Pathology and Cell
Biology, Columbia University, for providing human serum used in
the experiments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.electacta.2018.08.062.
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