Biosensors and Bioelectronics 110 (2018) 180–192

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

Recent advances in synthesis of three-dimensional porous graphene and its T

applications in construction of electrochemical (bio)sensors for small
biomolecules detection
⁎
Lu Lu
College of Food Science and Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: Electrochemical (bio)sensors have attracted much attention due to their high sensitivity, fast response time,
Electrochemical biosensors biocompatibility, low cost and easy miniaturization. Specially, ever-growing necessity and interest have given
Three-dimension rise to the fast development of electrochemical (bio)sensors for the detection of small biomolecules. They play
Porous graphene enormous roles in the life processes with various biological function, such as life signal transmission, genetic
Preparation
expression and metabolism. Moreover, their amount in body can be used as an indicator for diagnosis of many
Small biomolecules detection
diseases. For example, an abnormal concentration of blood glucose can indicate hyperglycemia or hypoglycemia.
Graphene (GR) shows great applications in electrochemical (bio)sensors. Compared with two-dimensional (2D)
GR that is inclined to stack together due to the strong π-π interaction, monolithic 3D porous GR has larger
specific area, superior mechanical strength, better stability, higher conductivity and electrocatalytic activity. So
they attracted more and increasing attention as sensing materials for small biomolecules. This review focuses on
the recent advances and strategies in the fabrication methods of 3D porous GR and the development of various
electrochemical (bio)sensors based on porous GR and its nanocomposites for the detection of small biomolecules.
The challenges and future efforts direction of high-performance electrochemical (bio)sensors based on 3D porous
GR for more sensitive analysis of small biomolecules are discussed and proposed. It will give readers an overall
understanding of their progress and provide some theoretical guidelines for their future efforts and development.

1. Introduction
Small biomolecules play enormous roles in the life processes and
various biochemical reactions, such as life signal transmission, genetic
expression and metabolism (Feng et al., 2014). The abnormal amount of
these small biomolecules in human body may cause various diseases.
For example, reactive oxygen species (ROS) mainly regulate DNA da-
mage and protein synthesis in cells. However, the excessive accumu-
lation of ROS in cells may cause cancer, cardiovascular and Alzheimer's
disease, etc. (Li et al., 2017). For another example, glucose participates
the metabolism processes and is a key biomolecule for providing energy
for human body. Nevertheless, more or insufficient glucose amount will
cause hyperglycemia or hypoglycemia (Zhang et al., 2016b). As a re-
sult, sensitive and rapid detection of the small biomolecule is tre-
mendously pivotal for early diagnosis and treatment for these diseases.
Compared with chromatography, spectrophotometry and fluorometry,
electrochemical techniques are more effective for sensitive, rapid and
real-time analysis towards these small biomolecules. Electrochemical
(bio)sensors have been attracted much attention and widely used due to
the good selectivity, high sensitivity, fast response time, biocompat-
ibility, low cost and easy miniaturization. And with the development of
nanotechnology, various electrochemical (bio)sensors based on nano-
materials have been constructed for determining small biomolecules.
Among these nanomaterials, graphene (GR) with a typical two-di-
mensional (2D) atomic structure has attracted enormous interest due to
its unique physicochemical properties, such as high electronic con-
ductivity (200 S m−1) (Stankovich et al., 2007), large specific surface
area (2630 m2 g−1) (Stoller et al., 2008), excellent thermal stability,
superior mechanical strength. In addition, GR also exhibits various
fascinating electrochemical properties for biosensing applications, in-
cluding wide electrochemical windows, high conductivity and electro-
chemical activity (Bahadır and Sezgintürk, 2016; Gao and Duan, 2015;
Han et al., 2014; Tung et al., 2017; Xu et al., 2017). However, the 2D
GR nanosheets easily aggregate together and the phenomenon is
usually irreversible, which can be attributed to the strong π-π stacking
and van der Waals interactions between the GR nanosheets (Kim et al.,
2010). Therefore, their application as electrode modifying materials
and electrocatalytical ability are limited for the detection of the small

⁎
Corresponding author.
E-mail address: lulu@qlu.edu.cn.

https://doi.org/10.1016/j.bios.2018.03.060
Received 18 January 2018; Received in revised form 11 March 2018; Accepted 27 March 2018
Available online 28 March 2018
0956-5663/ © 2018 Elsevier B.V. All rights reserved.
L. Lu
biomolecules. Many strategies have been tried to fabricate less ag-
gregated GR, such as carbon nanomaterials and GR composites, surface
functionalized GR, which have been used in the electrochemical (bio)
sensors (Bo et al., 2017).
Recently, three-dimensional (3D) porous GR materials have been
fabricated to increase the accessible real area and catalytical activity.
Due to the excellent characteristics for application, many strategies
have been designed to fabricate 3D porous GR, including self-assembly,
template-based method, chemical reduction, and chemical vapor de-
position (CVD) (Qiu et al., 2017). Compared with the common 2D GR
nanosheets, the 3D porous GR avoids the aggregation successfully, and
it exhibits more excellent characteristics, including larger inner space,
better mechanical stability (Donaldson, 2017) and higher catalytical
activity. These advantages of 3D porous GR provide conditions for the
applications in detecting small biomolecules. For example, Dong et al.
fabricated a macroporous GR foam (100–200 µm in pore size) with high
conductivity and it exhibited excellent sensing ability for selective and
sensitive determination of dopamine (DA) (Dong et al., 2012). Several
important reviews related to 3D GR have been published due to the fast
development of GR materials. Most of them focused on the fabrication
methods and main applications of 3D porous GR in energy conversion/
storage, environmental remediation (Wang et al., 2015), biofuel cells
(Qiu et al., 2017) and some biomolecules analysis (Bo et al., 2017).
Quiet recently, many studies on the fabrication of monolithic 3D porous
GR have been carried out for the application in electrochemical sensors.
And for the more sensitive sensing, efforts have been made to adjust the
pore size and morphology of the 3D GR (Qiu et al., 2015). However, the
fabrication of integrated 3D porous GR and the application for the
analysis of small biomolecules have not been dedicatedly and system-
atically summarized. As a result, it is very necessary to summarize and
review the recent development of the preparation methods of 3D
porous GR and its application for small biomolecules analysis and de-
tection.
The present review summarizes the recent advances of the fabri-
cation methods of monolithic 3D porous GR and focuses on their ap-
plications for the construction of electrochemical (bio)sensors for de-
tecting small biomolecules. The small biomolecules include glucose,
H2O2, DA, ascorbic acid (AA), uric acid (UA), DNA bases, epinephrine
(EP), metanephrine (MEP), levodopa (LD), adenosine triphosphate
(ATP), nitric oxide (NO), urea, methotrexate and melamine. Some in-
teraction between the structure of 3D GR and their performances for
sensing small biomolecules was discussed. Finally, the challenges and
the direction of future efforts associated with high-performance elec-
trochemical (bio)sensors based on 3D porous GR for more sensitive
analysis of small biomolecules are discussed and proposed.
2. Synthesis methods of 3D porous GR
2.1. Self-assembly strategies
2.1.1. Hydrothermal treatment
A typical example for the fabrication of 3D porous GR hydrogel
using hydrothermal reduction method was reported by Shi's group (Xu
et al., 2010). The graphene oxide (GO) sheets can be well dispersed via
the electrostatic interaction between the functional groups distributed
on the GO surface. Then, the oxygen functionalities disappeared with
the increase of the reduction time. As a result, the gradually increased
hydrophilicity of the reductive GO and π-π interaction between the
nanosheets made the GR sheets self-assembly grow to a conjugated
structure that was called a 3D porous GR hydrogel. The self-assembled
GR hydrogel had a good conductivity (5 × 10−3 S m−1), excellent
mechanical strength and thermal stability. Fig. 1A and B show the di-
gital photographs of the homogeneous GO suspension before and after
hydrothermal reduction, and the corresponding scanning electron mi-
croscope (SEM) image of the 3D porous GR hydrogel, respectively.
Fig. 1C shows the self-assembly process of porous GR during
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Biosensors and Bioelectronics 110 (2018) 180–192
hydrothermal reduction (Xu et al., 2010).
Using hydrothermal reduction method, Zhao et al. added pyrrole
(5 vol%) in the GO solution to prepare GR foam (Zhao et al., 2012).
During their preparation process, reductant and high-temperature
thermal treatment had to be applied to obtain 3D porous GR. Shi et al.
reported a simple, mild and clean approach to prepare a relatively or-
dered 3D GR through hydrothermal treatment without reductant and
severe thermal treatment (Shi et al., 2014). In their work, the pore size
of GR is from sub-micrometer to several micrometers, which is very
large and needs to be regulated to increase the specific surface area.
2.1.2. Chemical reduction
Recently, various reductants have been tried to assist the fabrication
of porous GR, including vitamin C (Sui et al., 2011), HI (Zhang and Shi,
2011), hydroquinone (Xu et al., 2013), sodium ascorbate (Chen and
Yan, 2011) and FeSO4 (Cong et al., 2012). As seen from these results,
the pore size of the porous GR was too large and usually distributed
randomly. Xie et al. reported a facile chemical reduction method to
fabricate porous GR hydrogel with relatively controllable pore size via
regulating the pH of the GO solution (Xie et al., 2016). In their work,
sodium ascorbate was used as the chemical reductant to reduce GO and
the porous GR hydrogel was obtained with changeable pore size.
2.1.3. Nanopore-etching method
The GR nanopores can be produced through etching process using
electron beam, photo, chemical or oxygen plasma-etching, which can
improve the properties of GR (Cao et al., 2015). A hydrothermally self-
assembled 3D GR foam with hierarchical pore arrangement by nano-
particle etching strategy was successfully fabricated (Zhao et al., 2014).
The new Fe2O3 etching GR material exhibited high electrocatalytic
activities for oxygen reduction reaction. The preparation process is
shown in Fig. 2A. The 3D GR gel was first prepared through hydro-
thermal treatment of GO solution with pyrrole (3 vol%). Then it was
immersed into a FeCl3 solution and heated to form Fe2O3 nanoparticles
and GR nanocomposites. Finally, Fe2O3 nanoparticles were etched by
hydrochloric acid, forming a 3D porous GR with small nanopores
(Fig. 2B). Recently, iron nitrate (Su et al., 2017) and CoOx (Lv et al.,
2017) were also used as the etching agents. The performances of these
as-prepared GR with the hierarchical pores (large GR pore and small
etching pores) via etching method were improved greatly, such as high
specific surface area (362 m2 g−1), high nanohole density (ca. 5.0 ×
109 holes per cm2), uniform pores size (2–50 nm) and excellent elec-
trocatalytic activity (Zhao et al., 2014).
It can be found that the self-assembly methods exhibited many ad-
vantages as follows. Firstly, self-assembly is a direct, facile and one-step
strategy to obtain 3D porous GR from 2D GR nanosheets. It not only
prevents the aggregation of 2D GR, but also constructs a monolithic and
interconnected 3D porous structure. From the electrochemical point of
view, the 3D porous GR with greatly increased conductivity, surface
area and mechanical strength, electrochemical activity can provide a
good modifier for electrochemical (bio)sensors (Duan et al., 2017).
Secondly, during the self-assembly process, the hydrophilic oxygen
functionalities on GR can encapsulate water molecules, which provides
an inherent biocompatible environment for the immobilization of bio-
molecules to construct electrochemical biosensors. Last but not the
least, the surface of 3D porous GR can be easily modified and functio-
nalized during the self-assembly and reduction processes (Zhang et al.,
2015), further improving the performance of electrochemical (bio)
sensors.
2.2. Templated methods
2.2.1. Template-based assembly
The morphology of the prepared materials usually base on that of
the templated materials and can be easily controlled by changing the
morphology of the templates. Choi et al. used polystyrene (PS) colloidal
L. Lu
spheres as the template to prepare 3D macroporous GR with con-
trollable pore size (Choi et al., 2012). Chemical reduction of GO was
finished with the mixture of PS. The hydrophobic GR was attached to
the PS spheres and through filtration treatment to obtain a thin film.
The PS template was then dissolved in toluene to form 3D GR structures
(Fig. 3). Almost in the same way, GO was reduced with the simulta-
neous removal of the PS templates during the ultrasonic spray pyrolysis
(Sohn et al., 2012). From the morphology of the GR, the pore size was
very large (~ 20 µm). Huang et al. used methyl functionalized silica
particles (30–120 nm) as the templates to fabricate lamellar-like GR
structures (Huang et al., 2012). Alumina fiber blanket was used as
another template. During the process, the GO/alumina fiber blanket
composites were annealed in N2 atmosphere at a high temperature
Fig. 2. (A) The fabrication process of N-doped GR mesh assembled foam. (B) SEM (a) and scanning transmission electron microscopy (STEM) (b) image of individual
sheet within N-doped GR mesh assembled foam. The inset in (b) is the enlarged TEM image of a single nanohole. (c) STEM image and C-, N- and O-elemental
mappings for a nanohole region of N-doped GR mesh assembled foam. Reprinted with permission from Zhao et al. (2014). Copyright 2014 The Royal Society of
Chemistry.
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Biosensors and Bioelectronics 110 (2018) 180–192
Fig. 1. (A) Photographs of a homogeneous GO aqueous
dispersion (2 mg/mL) before and after hydrothermal re-
duction at 180 °C for 12 h. (B) SEM image of the self-as-
sembled GR hydrogel interior microstructures. (C) The
proposed formation mechanism for self-assembled GR
hydrogel. Reprinted with permission from Xu et al. (2010).
Copyright 2010 American Chemical Society.
(500 ℃) and the fiber template was removed, forming a 3D GR with
microchannels (Yan et al., 2014). Fig. 4 shows the cartoon presentation
(A) of the preparation progress of microchannel-network graphene
foams and the corresponding SEM images (B and C) under different
magnifications. It is seen that the alumina fiber template can be easily
removed, resulting in an interconnected 3D continuous porous GR.
2.2.2. Chemical vapor deposition (CVD)
3D porous GR can be fabricated combining template and CVD
methods, during which the GR layer is directly grow on the templates
and a 3D bicontinuous porous GR structure is atomically formed. A
typical example is that 3D porous GR foam has been successfully pre-
pared using Ni foam as the template for the growth of GR layer (Chen
L. Lu Biosensors and Bioelectronics 110 (2018) 180–192

Fig. 3. (A) Schematic illustrating a procedure to fabricate 3D macroporous films through an embossing process using PS templates for embossed-chemically modified
graphene film. (B) Low-magnified and (C) high-magnified cross-sectional SEM images of embossed-chemically modified graphene film. (D and E) TEM images at
different magnifications of embossed-chemically modified graphene film. Reprinted with permission from Choi et al. (2012). Copyright 2012 American Chemical
Society.

et al., 2011). To avoid the collapse of the GR network, a poly(methyl
methacrylate) thin layer was deposited on the GR surface. Then a free-
standing 3D porous GR foam was finally obtained after the Ni foam
template and polymer layer were removed by hot HCl and acetone,
respectively. The specific surface area of this GR foam was about
850 m2 g−1, which was much higher than that fabricated using che-
mical reduction (100–700 m2 g−1) (Chen et al., 2011). However, the
pore size of the GR foam was very large and may affect their perfor-
mance. To decrease the pore size and obtain a bicontinuous porous GR,
the commercial Ni foam was replaced by nanoporous Ni as the template
(Ito et al., 2014). The nanoporous Ni template with a small pore size of
about 10 nm was fabricated by dealloying NiMn alloy (Qiu et al., 2014).
Then the pore and ligament size increased to 200–400 nm and a GR thin
film was deposited on the surface of the Ni ligament after the CVD
treatment. A 3D nanoporous GR with tubular-like ligaments was suc-
cessfully fabricated after removing the Ni template in HCl solution
(Fig. 5) (Ito et al., 2014) and its pore size can be adjusted from nan-
ometers to micrometers (Qiu et al., 2015). As reported, the smaller pore
and ligament size was favorable for the enhancement of the specific
surface area and the subsequent applications, such as biosensors and
energy storage. For the further increase of its performance, the 3D
porous GR can be decorated with many functional materials such as
metal nanoparticles (Qiu et al., 2013), metal oxides nanowires (Yue
et al., 2016) and polymers (Meng et al., 2016), etc.
Using templated methods, an obvious advantage is that the struc-
ture of the obtained 3D porous GR often highly ordered and monolithic
due to the regularity and integration of the templated materials. In
addition, the morphology of 3D porous GR can be easily controlled by
changing the morphology and structure of the templates (Huang et al.,
2012). From the electrochemical point of view, the morphology and
pore size can be easily adjusted according to the different electro-
chemical requirements. For example, the surface area can be further
enlarged, resulting in the increase of the electrochemical signal and the
amount of the immobilized biomolecules. And the 3D porous GR with
different pore size provides a monolithic substrate material for the
immobilization of different biomolecules.
Of course, both the self-assembly and templated methods are not
perfect. For example, the pore size of the 3D porous GR is large and the
reaction time is long using self-assembly methods. The 3D porous GR is
often not one-step prepared and the GR surface is usually hydrophobic
using templated methods (Qiu et al., 2017). However, the two methods
have their respective and obvious advantages for electrochemical ap-
plications and can be selected for different small biomolecules detec-
tion.

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L. Lu
Fig. 4. (A) Cartoon presentation of microchannel-network graphene foams preparation. (B and C) SEM images of microchannel-network graphene foams under
different magnifications. Reprinted with permission from Yan et al. (2014). Copyright 2014 The Royal Society of Chemistry.
3. Electrochemical (bio)sensors based on 3D porous GR and its
nanocomposites for small biomolecules
Monolithic 3D porous GR and its nanocomposites have been used to
construct advanced electrochemical (bio)sensors due to its unique
physicochemical properties, especially for their electrochemical prop-
erties such as high conductivity, wide electrochemical window and high
electrocatalytic activity resulting from the heteroatom doping or de-
fects (Qiu et al., 2015; Zhou et al., 2014, 2016). Here, the author in-
troduces and summarizes the electrochemical sensing applications of
monolithic 3D porous GR and its nanocomposites for small biomole-
cules.
3.1. Monolithic 3D porous GR
Compared with 2D GR nanosheets that easily aggregate, monolithic
3D porous GR has exhibited excellent sensing performance as the single
modifier to construct electrochemical (bio)sensors due to its higher
surface area, better electron transfer ability and mechanical strength,
etc. Therefore, it is necessary to introduce the electrochemical (bio)
sensors modified by monolithic 3D porous GR for the detection of small
biomolecules.
3.1.1. Glucose
Glucose is one of the most important small biomolecules and related
to the metabolism process closely. Sensitive and fast detection of glu-
cose in blood is pivotal for the clinical diagnosis of hypoglycemia or
diabetes. The performance of electrochemical biosensors based on
glucose oxidase (GOx) was greatly increased with the introduction of
3D porous GR. For example, a 3D interpenetrating porous GR was
electrodeposited on a glassy carbon electrode (GCE) and GOx was im-
mobilized on the modified GCE by coating the GOx and chitosan mixed
solution. Compared with the bare GCE, the direct electrochemistry of
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Biosensors and Bioelectronics 110 (2018) 180–192
GOx was achieved on the 3D interpenetrating porous GR/GCE with a
fast electron transfer rate constant (ks = 6.05 s−1). The porous GR/GOx
sensor had a good ability for glucose detection with a low detection
limit of 1.7 μM (S/N = 3) and a wide linear range of 0.02–3.2 mM. The
peak currents of the GOx/3D porous GR/GCE had no significant de-
crease after twenty repeated scans and one-month storage. The relative
standard deviation (RSD) of the peak current of five different modified
electrode was about 4%. The results showed that the biosensor had
good stability and reproducibility (Cui et al., 2013). Qian's group
achieved the direct electron transfer of GOx on 3D porous GR prepared
from ice-induced assembly (Qian and Lu, 2014; Wu et al., 2016). Fig. 6
shows the SEM images of GR-chitosan composite and porous GR (A and
D), and the electrochemical behaviors of GOx at the two porous GR
modified electrodes (B, C, E and F). It is seen that the two biosensors
fabricated using coating method detected glucose effectively and
showed good stability during ten-day storage.
3D porous GR can also be used for the fabrication of microelectrode
that is necessary for the in-vivo glucose detection. A new type of flower-
like porous GR wire microelectrode was successfully fabricated by di-
rect electrochemical assemble of graphene oxide on Pt wire with a
diameter of 250 µm. GOx was then immobilized on the porous GR by
adsorption and the GOx-based microbiosensor exhibited excellent di-
rect electron transfer properties and glucose sensing ability with good
linear ranges (8–148.0 and 148.0–430.6 μM) and detection limit (1 μM)
(Shi et al., 2015b). After continuous 100 scan cycles and one-month
storage, the electrochemical signal had no obvious change. The RSD of
the peak currents of five microsensors fabricated in the same way was
3.8%. The results indicated the biosensor had good stability and re-
producibility. The improved detection performances are attributed to
the increase of the amount of immobilized enzyme and the good elec-
tron transmission ability of porous GR. The analytical and detecting
performance towards glucose of the recent (bio)sensors based on
monolithic 3D porous GR are listed in Table 1, including operating
L. Lu
Fig. 5. Illustration of fabrication process of nanoporous GR. Reprinted with permission from Ito et al. (2014). Copyright (2014) Wiley-VCH Verlag GmbH.
potential, linear range, sensitivity and detection limit.
3.1.2. Hydrogen peroxide
As one of the most important intracellular biomolecules, H2O2 is a
typical representative of ROS and closely related to human's health (Bai
et al., 2016). Due to its long lift time, H2O2 molecule often penetrates
other cellular compartment and leads to some harmful biological da-
mage. Moreover, the amounts of H2O2 released from the living cells can
be considered as a newly emerged cancer biomarker (Wang et al.,
2016a). Therefore, it is very necessary and urgent to develop H2O2
sensors.
Table 2 summarizes the analytical performances of electrochemical
(bio)sensors in terms of operating potential, linear range, sensitivity
and detection limit for H2O2 detection. For electrochemical enzymatic
H2O2 sensors, monolithic 3D porous GR is an excellent matrix for im-
mobilizing enzyme due to its larger surface area, better biocompat-
ibility and higher electron conductivity (Zhou et al., 2014). Therefore,
horseradish peroxidase (HRP) was immobilized on porous GR prepared
using silver nanoparticles (AgNPs) etching method (Liu et al., 2016). In
their work, an AgNPs-GR nanocomposite was first prepared by hydro-
thermal synthetic strategy. Then the AgNPs were etched using 1.0 M
HNO3 solution, forming a porous GR structure. The electrochemical
H2O2 biosensor was constructed by dropping HRP/porous GR solution
on GCE surface and exhibited excellent performance for H2O2 detec-
tion, including a wide linear range (7 orders of magnitude) and a very
low detection limit (0.0267 nM). The authors tested the reproducibility
by measuring the current to 1 mM H2O2 using five independent
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Biosensors and Bioelectronics 110 (2018) 180–192
biosensors with RSD of 3.23%. And after 50 consecutive detection, the
current retained 85.14% of the original signal. For electrochemical
nonenzymatic detection, Wang et al. prepared a porous GR with very
small pores using Cu as the etching reagent and immobilized it on GCE
for H2O2 detection. Due to more edge defect sites and acidic groups, its
electrocatalytical activity and sensing performance were much better
than the non-porous GR (Wang et al., 2014a).
3.1.3. Dopamine (DA), ascorbic acid (AA) and uric acid (UA)
As the significant bioactive small molecules, DA, AA and UA play
crucial roles in the function of the metastasis, central nervous, renal and
hormonal systems. It is inspiring that many sensors based on 3D porous
GR have achieved simultaneous detection of DA, AA and UA. Table 3
summarizes the analytical performances of the recent electrochemical
sensors based on 3D porous GR and its composites for the AA, DA and
UA detection, including linear range, sensitivity and detection limit.
A monolithic 3D porous GR electrode was prepared using Ni foam as
the template and applied to detect DA, AA and UA simultaneously and
the detection limit for them was about 5 μM (Xiao et al., 2014). In
addition to Ni foam, PS with positive charge was also used as the
template. A 3D porous GR sensor was prepared by coating GR sus-
pension on a polished GCE (Wang et al., 2016b). The peak potentials of
AA (− 0.04 V), DA (+ 0.15 V) and UA (+ 0.3 V) (vs. SCE) are much
different from each other, which are the basis for the achievement of
the simultaneous detection. The currents of five 3D GR modified elec-
trodes were determined immediately and after seven-day storage, and
the sensor exhibited good reproducibility and stability. Another 3D
L. Lu Biosensors and Bioelectronics 110 (2018) 180–192

Fig. 6. (A) SEM images of the GR-chitosan composites on the substrate. (B) Cyclic voltammograms of the GOx/GR-chitosan/GCE (a) and GR-chitosan/GCE (b) in
nitrogen-saturated 0.1 M PBS (pH 7.0). Scan rate: 100 mV s−1. (C) Cyclic voltammograms of the GOx-Nafion/GR-chitosan/GCE in air-saturated 0.1 M PBS (pH 7.0)
with 0, 0.1, 0.4, 1.6 and 5.2 mM glucose. Scan rate: 10 mV s−1. Reprinted with permission from Qian and Lu (2014). Copyright 2014 The Royal Society of Chemistry.
(D) SEM images of PGR. Scale bar: 5 µm. (E) Cyclic voltammogram of the GOx/PGR/GCE in nitrogen-saturated 0.1 M PBS (pH 7.0). Scan rate: 100 mV s−1. (F)
Amperometric response of the GOx/PGR/GCE at 0.40 V in a stirring nitrogen-saturated 0.1 M PBS (pH 7.0). Reprinted with permission from Wang et al. (2014b).
Copyright 2014 Elsevier.

unmodified “as-grown” epitaxial GR with porous structures was directly
used for sensing DA, AA and UA detection (Kumar et al., 2015). As
reported, the vertically oriented GR provided fast electron transfer and
large electroactive area for sensing the active biomolecules. The peak
potentials of AA, DA and UA were demonstrated to be + 0.15, + 0.15
and + 0.3 V (vs. Ag/AgCl electrode) at the common GCE respectively.
When use 3D GR porous structure modified GCE, the peak potential of
AA was negatively shifted to − 0.055 V, indicating good electro-
catalysis towards AA oxidation. As a result, DA, AA and UA were si-
multaneously determined and the detection limit for UA was as low as
0.033 nM. To investigate the effect of porosity on the selectivity of GR,
numerical experiments and simulations have been carried out (Punckt
et al., 2014). The monolayer and porous GR film modified electrodes
were applied in detecting DA, AA and UA. It was demonstrated that the
porosity of GR provided strong apparent electrocatalytic effects and the
pore-size and pore-volume benefitted for separating the redox peaks of
DA, AA and UA, demonstrating the good electrochemical performance
of 3D porous GR.

Table 1
Analytical performances of electrochemical glucose (bio)sensors based on 3D porous GR.
Sensing material Operating potential/V (vs. Ag/AgCl) Linear range/μM Sensitivity/μA mM−1 cm−2 Detection limit/μM Ref.

GOx-PGR + 0.85a 0.25–27.5 755b 0.05 (He et al., 2017)

PGR/GOx + 0.4 140–8.160 16.3 8.7 (Wu et al., 2016)
chitosan-GOx-ERGO − 0.45a 20–3200 6.82 1.7 (Cui et al., 2013)
GOx/3D PGR-chitosan – 140–7000 11.2 17.5 (Qian and Lu, 2014)
GOx@flowers-shaped PGR – 8–148 267.34 1 (Shi et al., 2015b)
148–430.6 38.15
Ferrocene- chitosan/SWNTs/GOx/3D GR + 0.4 5–19,800 – 1.2 (Liu et al., 2014)
3D Ni/PGR – 103–104 400 – (Xiao et al., 2012)
Cu/3D GR hybrid + 0.60 − 3300 7880 0.018 (Hussain et al., 2015)
Co3O4 nanowires/3D PGR + 0.58 − 80 3390 0.025 (Dong et al., 2012)
Mn3O4/3D GR foam + 0.40 100–8000 360 10 (Si et al., 2012)
CuO/PrGO + 0.55 1–6000 207.3 0.5 (Zhao et al., 2015)
CuO/ammonia-doped-PrGO + 0.50 0.25–6000 1210 0.25 (Maaoui et al., 2017)
Ni(OH)2/3D GR foam + 0.55 1–1170 2650 0.34 (Zhan et al., 2014)
Cu(OH)2 nanorods/PGR + 0.60 1.2–6000 3360 1.2 (Shackery et al., 2016)
NiCo2O4/3D GR foam + 0.50 0. 5–590 2524 0.38 (Wu et al., 2015)
Hollow CuCo2O4 polyhedron/PrGO + 0.55a 0.5–3354 2426 0.15 (Yang et al., 2017)

PGR: porous graphene. ERGO: electrochemically reduced GO. SWNTs: single-walled nanotubes. PrGO: porous reduced graphene oxide.
a
Potential vs. SCE.
b
μA mM−1.

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Table 2
Analytical performances of electrochemical H2O2 (bio)sensors based on 3D porous GR.
Sensing material Operating potential/V (vs. Ag/AgCl) Linear range/μM Sensitivity/μA mM−1 cm−2 Detection limit/μM Ref.

−5 −5
HRP/PGR − 0.7 8 × 10 –0.664 – 2.67 × 10 (Liu et al., 2016)
2.77–835
b d
AuNPs-embedded PGR + 0.5 0.5–4900 75.9 0.1 (Qian et al., 2012)
Pt nanoflower/N-PGR − 0.25a 0.5–40,326 201.8 0.2 (Ren et al., 2016)
PtNPs/PGR − 0.1a 1000–1477 341.14 0.5 (Liu et al., 2015)
Pt-IL-PGR + 0.074a 10–4000 942.15 0.42 (Zhang et al., 2016a)
3D PGR/Cu2O − 0.3a 0.5–10,170 15.8d 0.2 (Ma et al., 2016)
3D N-doped PGR − 0.11a,ca,c 0.6–2125 1000 0.3 (Qi et al., 2017)
Pd/PDDA/PGR − 0.2a 2–1672 816.7 0.9 (Xue et al., 2016)
PtRu/3D GR foam + 0.32b,c 0–20 1023.1 0.04 (Kung et al., 2014)
PtAu/IL-ERGO − 0.25a 1.0–19,940 118 1.0 (Wang et al., 2016a)
Au nanoflowers/IL-GR foam − 0.6a 0.5–2300 425.6 0.1 (Zhang et al., 2017)
3D GR/polydopamine/thionine − 0.25a 0.4–660 169.7d ~0.08 (Xi et al., 2013)
3D-PGR − 0.4a 0.2–41,200 – 0.17 (Tian et al., 2017)
Pd/PGR − 0.2a 2–1672 57.7d 0.9 (Xue et al., 2016)

PtNPs: Pt nanoparticles. IL: ionic liquid. PDDA: poly(diallyldimethylammonium chloride).

a
H2O2 reduction potential.
b
H2O2 oxidation potential.
c
Potential vs. SCE.
d
μA mM−1.

3.2. Hybrid and doped 3D porous GR nanocomposites
To further increase the electrochemical sensing performance,
monolithic 3D porous GR is usually combined with other nanomaterials
to fabricate electrochemical (bio)sensors. And using hybrid and doped
3D porous GR nanocomposites as modifiers has become a development
trend for construction of electrochemical (bio)sensors for small bio-
molecules detection.
3.2.1. Glucose
The direct electron transfer of GOx has been further improved by
hybrid and doped 3D porous GR nanocomposites. A monolithic 3D GR
foam was used as the free-standing substrate for biofunctionalization by
one-step electrodeposition of GOx and single-walled carbon nanotubes
(SWNTs) doped ferrocene grafted chitosan matrix. Compared with the
common electrode, the biosensor exhibited good sensing ability with a
large linear range (5 µM to 19.8 mM), a low detection of 1.2 µM and
good storage stability and reproducibility. This attributed to its large
surface area, high conductivity and fast mass transport ability of 3D GR
foam and the synergistic effect of the nanocomposites (Liu et al., 2014).
Quiet recently, an electrochemical glucose biosensor was fabricated by
casting the GOx/porous GR mixture on GCE and then immersing in
HAuCl4 solution for the growth of gold nanoparticles (AuNPs) (He
et al., 2017). Compared with the bare GCE, an obvious direct electro-
chemical signal of GOx was observed at the nanocomposite and the
biosensor exhibited good detection performance for glucose with a
linear range of 0.25–27.5 µM and a detection limit of 0.05 µM (S/
N = 3).
For nonenzymatic sensing, 3D porous GR can be functionalized with
pure metal to form the nanocomposite for glucose detection. For ex-
ample, a 3D nanoporous GR encapsulated Ni core-shell electrode was
fabricated by annealing Ni@3D porous carbon at 750 ℃ for 20 min
(Xiao et al., 2012). The electrode exhibited good sensing ability for
glucose oxidation with a sensitivity of 400 μA mM−1 cm−2 and a linear
range of 1–10 mM. In addition, a Cu@3D porous GR hybrid electrode
was successfully fabricated for highly sensitive detection of glucose
(Hussain et al., 2015). In their work, the 3D GR was deposited on an Ni
foam (NF) by the CVD method and a thin Cu layer was evaporated on it,
resulting in hybrid Cu/3D GR/NF self-supporting electrochemical
sensor. It exhibited good electrocatalytic activity, selectivity and

Table 3
Analytical performances of electrochemical sensors based on 3D porous GR for AA, DA and UA detection.
Sensing materials Analyte Linear range/μM Sensitivity/μA μM−1 Detection limit/μM Ref.

Epitaxial graphene nanowall arrays AA 1.8–32 8.74a 1.8 (Kumar et al., 2015)
DA 0.02–2.6, 2.6–32 309.4, 22.7a 0.003
UA 0.02–2.6, 2.6–32 476.2, 19.4a 3.3 × 10−5

PGR/TiC AA 3–600 0.0557 1 (Fu et al., 2017)

DA 0.3–8, 8–60 4.208 0.1
UA 0.3–8, 8–60 2.655 0.1

3D PGR DA 0.2–8 2.104 0.2 (Wang et al., 2016b)

UA 1–60 292 1

PGR nanosheets DA 0.1–10, 10–40 – 0.001 (Gao et al., 2017)

UA 0.2–10, 10–50 0.003

3D honeycomb-like PGR DA 10−4–1.15 × 10−3 – 10−6 (Vilian et al., 2016)

3D N-GR skeleton DA 3–100 9.87a 0.001 (Feng et al., 2015)
flower-like RGO DA 5–600 – 3 (Wang et al., 2014b)
Fe2O3 NPs/PGR/polyimide-based carbon aerogel DA 5–500 – 0.109 (Zhang et al., 2016c)
PGO/AuNPs/PGO DA 0.1–30 – 1.28 (Choo et al., 2017)
3D-rGO DA 5–1000 – 0.17 (Yu et al., 2014)

PGO porous graphene oxide.

a
μA μM−1 cm−2.

187
L. Lu
excellent cycling stability for glucose sensing and the detection limit
was as low as 18 nM (S/N = 8.6). The authors indicated that the Cu/3D
GR hybrid can be a promising material for sensing glucose in real ap-
plications due to its superior long cycling stability and so high sensi-
bility.
The metal oxides and hydroxides have been used to be constituted
with 3D porous GR for constructing nonenzymatic glucose sensors.
Zhang's group in situ synthesized cobalt oxide (Co3O4) nanowires on 3D
porous GR foam to construct a monolithic free-standing electrode for
nonenzymatic electrochemical glucose detection (Dong et al., 2012).
The structure and morphology of 3D porous GR/Co3O4 nanocomposite
was characterized exhaustively. The SEM images of 3D porous GR foam
(A), GR/Co3O4 (B) and the glucose sensing behaviors (C and D) are
shown in Fig. 7. Without 3D porous GR, the Co3O4 nanowires/GCE
showed narrower cyclic voltammetry profile and less sensitive elec-
trochemical signal, indicating the importance of 3D porous GR foam
substrate. The authors demonstrated that the sensor detected glucose
with a much high sensitivity of 3.39 mA mM−1 cm−2 and a detection
limit lower than 25 nM (S/N = 8.5). Moreover, the 3D GR/Co3O4
electrode stayed stable after 500 scanning cycles. As another example,
CuO nanoparticles were anchored on porous GR using hydrothermal
method (Zhao et al., 2015). The hybrid electrochemical sensor was
fabricated by dip-coating a homogenous suspension of CuO/porous GR
and Nafion (10%) on a polished GCE. Compared with the non-porous
GR/CuO/GCE, the porous GR/CuO/GCE had an enhanced electro-
catalytical ability for glucose direct oxidation with a wide linear range
of 0.001–6 mM, a low detection limit of 0.5 μM and excellent precision
for glucose detection in human serum. The reproducibility was eval-
uated through detecting eight glucose sample and the RSD was 4.4%.
The current was kept about 82% of the original signal after scanning for
3500 s, indicating its good stability for long-time detection (Zhao et al.,
2015). Quite recently, CuO nanoparticles (CuO NPs) were supported on
3D porous ammonia-doped GR by electrophoretic deposition, forming
3D porous ammonia-doped GR/CuO NPs composites (Maaoui et al.,
2017). The nanocomposites/gold electrode exhibited good
188
Biosensors and Bioelectronics 110 (2018) 180–192
Fig. 7. SEM images of (A) 3D porous GR foam
and (B) 3D GR/Co3O4 nanowire composite. (C)
Cyclic voltammogram of GR/Co3O4 modified
electrode in the presence of various con-
centrations of glucose (0, 0.2, 0.4, 0.6, 0.8, and
1 mM). Scan rate: 20 mV s−1. (D)
Amperometric response of the sensor to glu-
cose at the potential of 0.58 V. Reprinted with
permission from Dong et al. (2012). Copyright
2012 American Chemical Society.
amperometric detection ability of glucose with a high sensitivity of
1210 μA mM−1 cm−2 and a detection limit of 0.25 μM (S/N = 3) and
had a long-term stability and good reproducibility. The authors in-
dicated the much higher sensitivity of their electrochemical sensor for
glucose than non-porous GR sensors, which was attributed to the 3D
structure of the hybrid material. A flower-like Cu(OH)2 nanorods
modified 3D porous GR was fabricated for electrochemical detection of
glucose (Shackery et al., 2016). The porous GR foam was first attached
to glass microslide and the Cu(OH)2 nanorods was deposited on it via a
facile and non-expensive chemical bath deposition method, forming a
hybrid electrochemical sensor. The Cu(OH)2 nanorods with a diameter
of about 100 nm were well covered on the porous GR structure, which
had a large surface area (9.44 cm2). Thus the 3D nanocomposite in-
creased the sensitivity (3360 μA mM−1 cm−2) of the glucose sensor
greatly. The reproducibility of the sensor was evaluated by five mea-
surements and the RSD was 5.2%. The current decayed by 9.1% during
twenty-time measurements within two months, showing good stability.
3.2.2. Hydrogen peroxide
Monolithic 3D porous GR have been well functionalized with me-
tals, metal oxides, alloys and polymer to form nanocomposites for
constructing electrochemical H2O2 sensors. The analytical perfor-
mances of electrochemical sensors based on them are listed in Table 2.
Guo's group successfully fabricated various porous GR using CaCO3 (Liu
et al., 2015) and ZnO (Xue et al., 2016) as the templates. The synergetic
porous GR and Pt or Pd nanoparticles greatly improved the detection
performances of the sensors. Take Pt/porous GR nanocomposite for
example, the electrochemical sensor was prepared by casting Pt/GR
suspension on the GCE surface and dried in air. The illustration of the
preparation process of Pt/porous GR samples and its sensing behaviors
for the H2O2 reduction are shown in Fig. 8. It can be seen that sensor
detected H2O2 with a wide linear range of 1–1477 μM, a high sensitivity
of 341.14 μA mM−1 cm−2 and a low detection limit of 0.50 μM.
Moreover, the authors indicated that it exhibited excellent selectivity,
long-term storage stability and reproducibility (Liu et al., 2015). Based
L. Lu
Fig. 8. (A) Illustration of the preparation process of Pt/porous GR samples. (B) Cyclic voltammograms of Pt/porous GR/Nafion/GCE in 0.1 M PBS (pH 7.0) containing
0 and 5 mM H2O2. (C) Amperometric curve of Pt/porous GR/Nafion/GCE with successive additions of H2O2 at − 0.1 V. Inset: the amperometric response with
successive addition of H2O2 with lower concentration. Reprinted with permission from Liu et al. (2015). Copyright 2015 Elsevier.
on the similar strategy, Pt nanoflowers (Ren et al., 2016), Au nano-
flowers (Zhang et al., 2017) and Fe3O4 quantum dots (Zhao et al., 2017)
were decorated on N-doped porous GR and ionic liquid functionalized
GR framework and 3D GR, respectively. These porous GR had very
different morphologies, including nanopores in single layer GR and
multilayer assembled pores among GR. These nanohybrids-based elec-
trochemical sensors exhibited excellent performance for H2O2 detec-
tion. Using polymer as the modifier, a monolithic 3D porous GR pre-
pared via CVD method and functionalized with thionine through the
linker polydopamine (Xi et al., 2013). The constructed sensor detected
H2O2 with a wide linear range (0.4–660 μM), a low detection limit
(80 nM), high sensitivity (169.7 μA mM−1), good stability and re-
producibility. The response to 20 μM H2O2 retained 93.6% of the ori-
ginal signal after seven-day storage and the RSD was 2.1% (n = 7). The
reproducibility of three independent sensors to 20 μM H2O2 was tested
and the RSD was 3.2% (n = 3).
It is worth noting that the amount of H2O2 in cells is a key indicator
to estimate the physical condition of cells and the human body. It is
inspiring that many electrochemical sensors based on hybrid 3D porous
GR nanomaterials have been applied for real-time monitoring and in
situ detecting H2O2 released from living cells successfully (Liu et al.,
2015; Tian et al., 2017; Xi et al., 2013; Zhang et al., 2017; Zhao et al.,
2017), benefiting from their good detection performances. The regular
sized electrochemical H2O2 sensors can hardly realize the measurement
of mini volumes from body fluid for in vitro clinical analysis. Quite
recently, Wang et al. designed a novel hierarchical nanohybrid micro-
sensor based on PtAu nanoflwers/3D porous GR-wrapped activated
189
Biosensors and Bioelectronics 110 (2018) 180–192
carbon fiber (Wang et al., 2016a). The nanocomposite-based micro-
sensor exhibited high sensitivity (118 μA mM−1 cm−2), wide linear
range (1 μM to 19.94 mM), low detection limit (1.0 μM) for real-time
tracking H2O2 released from normal and cancer cells. The mechanical
stability, long-term stability and reproducibility of the H2O2 micro-
sensor were evaluated in detail and the results were satisfying. The
microsensor can evaluate the therapeutic activity of the antitumor
drugs through in vivo H2O2 detection, providing a significant indicator
for cancer diagnosis and management.
3.2.3. DA, AA and UA
3D porous GR has been combined with other nanomaterials to
construct electrochemical sensors to further improve the detection
performance and reduce the adsorption of DA, AA and UA on electrode.
For example, 3D GR foam decorated with vertically aligned ZnO na-
nowire arrays (NWA) was applied to selectively detect DA, AA and UA
at separate potentials (Fig. 9) (Yue et al., 2014). The high selectivity
was ascribed to gap difference between the highest occupied and lowest
unoccupied molecular orbital of biomolecules. The authors concluded
the ZnO NWA/GR foam nanohybrid improved the selectivity, sensi-
tivity and stability of the sensor. During the real application, it was
important to find that the UA level was 25% lower in Parkinson's dis-
ease patient than in healthy human, indicating that UA was able to be
used as a biomarker for Parinson's disease. Fe2O3 nanoparticles in situ
grew on highly porous GR/polyimide carbon aerogel to form a nano-
composite (Zhang et al., 2016c). The nanocomposite-based sensor can
effectively simultaneous detection of DA, AA and UA. For DA, the
L. Lu
Fig. 9. (A) Schematic of NWA/GF electrode and detection of AA, DA and UA. (B-C) SEM images of the ZnO NWA on the 3D GF at different magnifications. (D–F) DPV
curves for UA, DA and AA at different concentrations using a ZnO NWA/GF electrode. Concentrations of UA and DA: 0, 0.001, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 0.75
and 1 μM. Concentrations of AA: 0, 1, 2.5, 5, 7.5 and 10 μM. Insets: plots of the oxidation peak current vs. concentration of each biomolecule. Reprinted with
permission from Yue et al. (2014). Copyright 2014 American Chemical Society.
sensor exhibited a very low detection limit of 0.109 μM and a linear
range of 5–500 μM.
3.2.4. DNA bases
As the composition of two complementary base pairs, adenine (A),
thymine (T), guanine (G) and cytosine (C) are closely related to the
stability of DNA, genetic information and DNA replication (Watson and
Crick, 1953). The deficiency or mutation of the immunity is usually
attributed to the abnormal changes of these bases, which may induce
some diseases such as cysticfibrosis and Alzheimer's disease (Valko
et al., 2004). Therefore, sensitive and accurate detection of DNA bases
is very imperious and necessary.
Li et al. fabricated 3D porous N-doped GR using a simple, eco-
friendly and low-cost strategy that combined the hydrothermal as-
sembly and freeze-drying method without any template and other
support framework (Gao et al., 2017). Their characterization showed
the cross-links of 3D porous structure (pore size 3.2–8.5 µm) and high
electroactive area. Compared with 2D GR, the 3D porous GR exhibited
more excellent performance for sensing adenine with low concentration
ranging from 0.02 to 1.2 μM and a detection limit of 8 nM. The RSD of
the responses to 1.0 μM adenine obtained at a same 3D N-doped GR
sensor was 2.17% (n = 6), indicating its good repeatability. The re-
sponses of six independent sensors were tested immediately and after a
month storage, exhibiting good reproducibility and stability. The au-
thors attributed these excellent sensing performances to the extra-
ordinary electron transfer properties and the enrichment of porous GR
for adenine (Li et al., 2016).
The simultaneous determination of DNA bases (A, T, G and C) is
much more necessary. Quiet recently, Sun et al. successfully fabricated
an electrochemical sensor using Cu-N-doped GR with ultralight and
porous structure as the sensing matrix via coating method (Sun et al.,
2017). It was amazing that the electrochemical detection potentials of
A, T, G and C separated absolutely with each other. Their oxidation
peak potentials on the sensor were about + 0.87, + 1.05, + 0.59 and
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Biosensors and Bioelectronics 110 (2018) 180–192
+ 1.30 V (vs. Ag/AgCl), respectively, and the detection concentration
ranges were 0.37–5.18 μM, 198.2–5551 μM, 0.132–6.62 μM and
270.0–1575 μM, respectively. The Cu-N-doped porous GR/GCE also
exhibited good stability during four-week storage and good reprodu-
cibility with RSD < 5%. The simultaneous detection of A, T, G and C
demonstrated that the excellent sensor based on 3D porous GR can be
applied in the clinical medicine and early diagnosis of some diseases.
The analytical performances of the electrochemical sensors based on 3D
porous GR and its composites for DNA bases detection are summarized
in Table 4, including linear range, sensitivity and detection limit.
3.2.5. Other small biomolecules
In addition to the above common biomolecules, other important
small biomolecules also play great roles in lives. EP is an important
catecholamine neurotransmitter and plays a key role in central nervous
system. For sensing EP, a N-doped 3D porous GR modified electrode
was fabricated and electrochemically characterized using Ru(NH3)6Cl3
as the probe (Yang et al., 2015). The redox peak currents were much
higher than that on bare GCE, attributing to the excellent properties of
N-doped porous GR. It is interesting that the sensor can simultaneously
detect the EP and MEP, which is a metabolite of EP during the inter-
action with transferase. The detection potentials were about + 0.14
and + 0.47 V (vs. SCE). The linear ranges for EP and MEP were 1.0 μM
to 1.0 mM and 1.5 μM to 0.41 mM, and the detect limits were 0.17 μM
and 1.3 μM, respectively. After parallel measurements, independent
detection and storage experiment, the N-doped porous GR sensor
showed good repeatability, reproducibility and stability.
LD is the immediate precursor of DA and vital for DA synthesis.
Though the amount of DA is not enough in body, DA cannot be used as
a medicine for direct injection due to it fails to pass the blood-brain
barrier. But LD can realize this aim (Dauer and Przedborski, 2003). A
ZnO nanowire arrays (NWAs) modified 3D porous GR obtained by CVD
using Ni foam as the template was successfully applied in the LD de-
tection (Yue et al., 2016). In presence of UA, the sensor can effectively
L. Lu Biosensors and Bioelectronics 110 (2018) 180–192

Table 4
Analytical performances of electrochemical sensors based on 3D porous GR for the detection of DNA bases and other small biomolecules.
Sensing materials Analyte Linear range/μM Sensitivity/μA μM−1 Detection limit/μM Ref.

PGR nanosheets G 0.7–100, 100–2000 – 0.29 (Gao et al., 2017)

A 0.15–10, 10–50 – 0.002

3D N-doped GR A 0.02–1.2 6.8954 0.008 (Li et al., 2016)

GR

Cu-N-doped RGO A 0.37–5.2 3.506a 0.02 (Sun et al., 2017)

T 198–5551 7.305a 0.008
G 0.132–6.6 0.03292a 9
C 270.0–1575 0.05673a 10

N-doped 3D PGR/chitosan EP 1–1000 0.021 0.67 (Yang et al., 2015)

MEP 1.5–410 0.0095 1.3

ZnO NWAs/3D GR foam LD 0.05–20 3.15 0.05 (Yue et al., 2016)

3D porous GR/gold ATP 0.0002–0.5 – 10−4 (Shi et al., 2015a)
Au NPs/3D GR NO 0.2–6 – 0.009 (Li et al., 2015)
NiCo2O4/3D GR urea 60–300 166b 5 (Nguyen et al., 2016)
3D GR-CNT methotrexate 0.7–100 – 0.07 (Asadian et al., 2017)
3D porous GR-porous PdCu NPs melamine 0.01–1 – 0.002 (Lei et al., 2014)

ZnO NWAs/3DGF: ZnO nanowire arrays/three-dimensional graphene foam.

a
μA mL μg−1.
b
μA mM−1 cm−2.

determine LD with a sensitivity of 3.15 μA μM−1, a linear range of
0.05–20 μM and a detection limit of 50 nM and showed good selectivity,
stability and reproducibility. The authors believed that this good per-
formance was attributed to the excellent electrocatalytic activity, con-
ductivity and high electroactive area from the combination of the ad-
vantages of ZnO NWAs and porous GR.
As the “energy currency” in cells, a small linear biomolecule ATP
plays great roles in energy storage, transfer and nucleic acid synthesis.
Shi et al. (2015a) fabricated a 3D porous GR film on gold substrate by
hydrothermal reduction and constructed an electrochemical aptasensor
for detecting ATP through the immobilization of ATP aptamer probe
and competing reaction between Ferrocene and ATP. The authors at-
tributed the high selectivity, stability and sensitivity of the sensor to the
large surface area, high conductivity and excellent binding strength of
the 3D porous GR.
NO plays many important roles in neurotransmission, vasorelaxa-
tion, immune defense function and pathogenesis of some diseases. A
gold nanoparticles and 3D porous GR nanocomposite was prepared and
used to construct an electrochemical NO sensor. The sensor had a
sensitive detection for NO with fast response, a linear range of 0.2–6 μM
and a low detection limit of 9 nM (S/N = 3). It was also used for in situ
detection of NO released from living cells, providing a good strategy for
distinguish normal and cancer cells (Li et al., 2015).
Urea is a metabolic end product in body. The amount of urea in
urine can evaluate many metabolic disorders, such as renal function.
For sensitive detection of urea, Yoon's group synthesized a bimetallic
NiCo2O4 on 3D porous GR (Nguyen et al., 2016). The electrochemical
urea sensor was constructed by depositing the NiCo2O4/3D GR nano-
composite on indium tin oxide (ITO) glass and dried in air. It exhibited
a high sensitivity of 166 A mM−1 cm−2, a linear range of 0.06–0.3 mM
and a detection limit of 5.0 μM towards urea. The sensor showed high
stability with a little decrease of the sensitivity (only 5.5%) after four-
month storage. It was amazing that the response time for urea was as
fast as about 1.0 s, demonstrating the sensor was excellent for sensing
urea in urine samples.
The electrochemical sensors based on 3D porous GR and its nano-
composites can also sensitively detect methotrexate (Asadian et al.,
2017) and melamine (Lei et al., 2014), which are not exist in human
body but can be found in blood when they are eaten and metabolized.
The analytical performances of the recent electrochemical sensors
based on 3D porous GR and its nanocomposites for the detection of
these small biomolecules are summarized in Table 4.
4. Conclusions and outlook
The design strategies and fabrication methods of 3D porous GR with
different morphology and structures have been comprehensively sum-
marized. Electrochemical (bio)sensors based on 3D porous GR and its
nanocomposites have shown excellent performances for the detection of
small biomolecules, including high sensitivity, good selectivity, wide
linear ranges and low detect limits. They are attributed to the unique
properties of 3D porous GR that combines the advantages of 2D and 3D
nanomaterials. Although these achievements deserve much con-
gratulations, many challenges and opportunities still remain in this
area.
(1) It is very necessary to precisely control the surface morphology and
structure of 3D porous GR, including pore size, pore volume and
surface properties. The larger surface area is favorable to enhance
the detective signal for small biomolecules. Integrated 3D GR with
hierarchical and controlled pores should be paid much attention to
due to the easier achievement for the immobilization of bioma-
cromolecules and detection of small biomolecules.
(2) The further increase of the electrocatalytic activity of 3D porous GR
will enhance the performance of electrochemical (bio)sensors for
small biomolecules. This requires more active sites that can be
realized through increasing surface area and designing controlled
doping GR with other atoms.
(3) The electrocatalytic mechanisms of 3D porous GR for small bio-
molecules and the relationship between the GR structure and the
catalytic performance are still required to be investigated in the
future. The mechanisms and theories will guide the design and
fabrication of porous GR with higher electrocatalytic activity.
(4) It is imperative to achieve the real applications of the electro-
chemical (bio)sensors for small biomolecules in biological samples,
which are limited by the much low concentration and many inter-
feres in real biological systems. Therefore, great efforts are required
to be made to improve the sensitivity and selectivity of the (bio)
sensors. Moreover, microsensors based on miniaturized 3D porous
GR are necessary to be designed and applied in vivo analysis.
Finally, it is worth believing that through concerted efforts of our
researchers, novel 3D porous GR with more fascinating properties will
be designed and fabricated, and the electrochemical (bio)sensors based
on the novel porous GR and its nanocomposites will exhibit more

191
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remarkable analytic performance for small biomolecules.
Acknowledgements
This work was supported by the special funds for Taishan Scholars,
the Scientific Research Foundation of Qilu University of Technology
(0412048489), Natural Science Research Key Project of Anhui
Provincial Department of Education (KJ2016A860) and National
Natural Science Foundation of China (31771933).
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