Trends in Analytical Chemistry 91 (2017) 53e66

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Trends in Analytical Chemistry

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Graphene based sensors and biosensors

Celine I.L. Justino a, b, *, Ana R. Gomes a, Ana C. Freitas c, Armando C. Duarte a,
Teresa A.P. Rocha-Santos a
a
Department of Chemistry & CESAM, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal
b
ISEIT/Viseu, Instituto Piaget, Estrada do Alto do Gaio, Galifonge, 3515-776 Lordosa, Viseu, Portugal
c
Universidade Cato
lica Portuguesa, CBQF e Centro de Biotecnologia e Química Fina, Laborato
rio Associado, Escola Superior de Biotecnologia, Rua Arquiteto
Loba~o Vital, Apartado 2511, 4202-401 Porto, Portugal

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

Article history: Graphene has contributed to the fabrication of sensitive sensors and biosensors due to its physical and
Available online 10 April 2017 electrochemical properties. This review discusses the role of graphene and graphene related materials for
the improvement of the analytical performance of sensors and biosensors. This paper also provides an
Keywords: overview of recent graphene based sensors and biosensors (2012e2016), comparing their analytical
Analytical performance performance for application in clinical, environmental, and food sciences research, and comments on
Biosensor
future and interesting research trends in this field.
Graphene
© 2017 Elsevier B.V. All rights reserved.
Graphene oxide
Sensor

1. Introduction
In the latest years, the construction of sensors and biosensors
has been improved with the incorporation of various nanomaterials
such as nanowires synthesized from metals (e.g., Ni, Cu, Au, Pt),
metal oxides (ZnO, SnO2, Fe2O3), and silicon/indium/gallium
semiconductors (Si, InP, GaN), quantum dots based on CdSe, CdTe or
CdSeTe, carbon nanotubes (CNT), and metal nanoparticles (based
on Au, Cu, Pd, Co, Ag, or Pt) due to their conductive properties, high
surface-to-volume ratio, and good biocompatibility, thus contrib-
uting to the improvement of the analytical performance of such
sensors and biosensors [1e3]. Recently, graphene has dominated
the scientific interest in order to compete with such nanomaterials
to the fabrication of sensors and biosensors.
Graphene is a planar sheet of carbon atoms and it is the basic
structural element of graphite, CNT, and fullerenes. The carbon
bonds are sp2 hybridized, where the in-plane sCeC bond is one of
the strongest bonds in materials and the out-of-plane p bond is
responsible for the electron conduction of graphene, providing the
weak interaction among graphene layers or between graphene and
substrate [4]. Such electron configuration is the basis to the large
surface area of graphene (2630 m2 g
1), which is higher than that of
graphite (10 m2 g
1) and CNT (1315 m2 g
1), high mechanical
* Corresponding author. Department of Chemistry & CESAM, University of Aveiro,
Campus de Santiago, 3810-193 Aveiro, Portugal. Fax: þ351 232 910 183.
E-mail address: celinejustino@ua.pt (C.I.L. Justino).
strength (200 times greater than steel), high electrical conductivity,
which is 60-fold greater than single-walled CNT (SWCNT), and six
orders of magnitude higher than copper, and high elasticity and
thermal conductivity [5e7].
Other related materials, which belong to the graphene family,
are the graphene oxide (GO) and the reduced GO (rGO). GO is
layered and oxygenated graphene sheets that contain oxygen
functional groups, such as epoxides, carboxyls, hydroxyls, and al-
cohols, located on the edge and surface of the sheet with a carbon
to oxygen ratio of approximately 3:1 [8]. The presence of oxygen
functional groups on the graphene surface may enhance the het-
erogeneous electron and charge transfer rate and make GO water-
soluble and biocompatible [9]. After reduction, GO transforms into
rGO with some residual oxygen and structural defects, yielding
high thermal conductivity comparable to that of doped conductive
polymers, about 36 times higher than that of Si and about 100 times
higher than that of GaAs [10].
Due to its planar shape and chemical structure, the graphene
has various advantageous properties in comparison to other ma-
terials to the construction of sensors and biosensors. For example,
the atomic thickness of the graphene sheet and its extremely high
surface-to-volume ratio make such material highly sensitive to-
wards the change of local environment conditions, which is an
important advantage in the sensing field, since all carbon atoms
interact directly with the analytes, thus promoting higher sensi-
tivity than silicon nanowires or CNT, thus contributing to the
fabrication of improved electrochemical sensors and biosensors

http://dx.doi.org/10.1016/j.trac.2017.04.003
0165-9936/© 2017 Elsevier B.V. All rights reserved.
54 C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66
[11,12]. In comparison to CNT, graphene has two main advantages
regarding its application in electrochemical sensors and biosensors,
that is, graphene does not contain metallic impurities, which
interfere with the material electrochemistry, and the production of
graphene used graphite, which is cheap and accessible [12]. In
addition, graphene can be easily connected to biomolecules by pep
stacking and hydrophobic interactions and the lower noise ratio,
facile functionalization, solution-processibility, and biocompati-
bility are advantages of graphene as channel material [9,11].
The graphene functionalization can be achieved with: a) poly-
mers, from their in situ synthesis obtaining high-strength and
conductive materials; for example, the GO/polyaniline (PANi)
composites can be prepared by the in situ polymerization of aniline
in GO dispersion; b) nanoparticles, which immobilization in gra-
phene occurs through hydrophobic and static interactions and
promotes high conductivity and excellent thermal stability [7]. The
fabrication of graphene/nanoparticles composites is commonly
based on in situ growth and solution mixing by reducing metal ions
since the oxygenated groups on the surfaces of both GO and gra-
phene facilitate the nucleation of metal nanoparticles; and, c)
biomolecules such as DNA, proteins, peptides, and cellulose, which
can also be incorporated in graphene surface through physical
adsorption or chemical bonding, modifying its structure due to the
various surface functional groups of such biomolecules [7]. In the
latest five years, various papers reviewed the graphene based
sensors and biosensors in different perspectives, and directed to
general sensors and biosensors [13e16], electrochemical sensors
and biosensors [5,6,11,12,17e19], and optical sensors [9,20], and for
the detection of biomarkers [5], heavy metals [8], and glucose [21].
In the present review, the literature concerning the construction
of sensors and biosensors based on graphene and graphene related
materials are critically updated, covering the period of 2012e2016,
for the detection of various analytes reported in clinical, environ-
mental, and food research areas. The analytical performance of
graphene based sensors and biosensors and their advantages and
limitations are compared, and some approaches are also reported
in order to improve their analytical performance.
2. Graphene and graphene related materials based sensors
and biosensors
In the latest years, a large number of sensors and biosensors based
on graphene and graphene related materials (GO and rGO) have been
reported in various areas such as clinical, environmental, and food
sciences research. Although the majority of sensors and biosensors
has an electrochemical transduction principle, optical and piezo-
electric sensors and biosensors have been also fabricated, as shown
in the following subsections. The analytical performance of sensors
and biosensors has been evaluated through various analytical figures
of merit such as sensitivity, limit of detection, repeatability, and
reproducibility, which are commonly used to validate an analytical
method, ensuring the quality of the results [22]. The transduction
mechanism and the morphological characteristics of sensors can
influence their sensitivity and limit of detection and such figures of
merit should be evaluated in order to assess, respectively, the
analytical reliability and capacity of sensors [22].
2.1. Graphene
Sensors and biosensors with graphene have been used in: a)
clinical applications, for example, to the detection of glucose,
cholesterol, H2O2, dopamine, ascorbic acid, uric acid, CEA, a-feto-
protein, thrombin, and PSA; b) environmental applications, for
example, to the detection of metal ions such as Cd2þ and Pb2þ,
pesticides such as methyl parathion, chlorpyrifos, and carbofuran,
as well as hydrogen; and, c) food applications, for example, to the
detection of erythromycin, tryptamine, and Staphylococcus aureus.
The combination of graphene sheets with polyvinylpyrrolidone
(PVP) [23] or nanoparticles [24e26] can enhance the analytical
response of sensors and biosensors, mainly improving their sensi-
tivity [24,27], limit of detection [27], and reproducibility [27].
Table 1 shows the analytical parameters of sensors and biosensors
fabricated with graphene and the analytical performance of some
examples listed in Table 1 is also discussed and compared in this
subsection.
An electrochemical sensor based on glassy carbon electrode
(GCE) with copper nanoparticles and graphene was proposed to the
detection of glucose [25]. The combination of such nanomaterials
provided better analytical response than the same sensors without
graphene, that is, the electrochemical sensor also exhibited much
better electrocatalytic properties for glucose oxidation when
compared to sensor only with copper nanoparticles, which is
attributed to the large surface area, high conductivity, and fast
electron transfer provided by graphene sheets.
To the detection of cholesterol, a graphene electrochemical
enzymatic biosensor onto graphite electrodes [28] and an
electrochemical enzymatic biosensor based on a plati-
numepalladiumechitosanegraphene hybrid nanocomposite onto
GCE [29] were fabricated. The analytical performance of the gra-
phene hybrid nanocomposite biosensor (limit of detection of
0.75 mM and linear range between 2.2 and 520 mM) [29] was better
than the graphene biosensor (limit of detection of 5 mM and linear
range between 50 and 300 mM) [28]. The presence of the hybrid
nanocomposite together with the unique catalytic activity and
chemical selectivity of the bimetallic nanoparticles not only could
accelerate the direct electron transfer from the redox enzyme to the
electrode surface, but also could enhance the immobilized amount
of cholesterol oxidase, thus enhancing its electrochemical proper-
ties and consequently its analytical performance for the detection
of cholesterol. The construction of low-cost, simple, and sensitive
optical enzymatic biosensors based on electrogenerated chem-
iluminescence principle, where cerium oxide and graphene com-
posite were immobilized onto GCE, was reported [30]. A
comparative study has been done by using three kinds of bio-
sensors based on cholesterol oxidaseecerium oxideeGCE, choles-
terol oxidaseegrapheneeGCE, and cholesterol oxidaseecerium
oxideegrapheneeGCE, as shown in Fig. 1A. The electrogenerated
chemiluminescence intensity of luminol has been enhanced to a
much greater extent in the presence of cerium oxideegraphene,
which is attributed to its electrocatalytic properties since more
efficient active sites have been created for the catalytic oxidation of
H2O2 to produce various reactive oxygen species that can enhance
the chemiluminescence intensity of luminol.
Recently, a paper-based cholesterol biosensor was proposed
using a novel nanocomposite of grapheneePVPePANi, verifying
that the presence of small amount of PVP (2 mg mL
1) in the
nanocomposites can substantially improve the dispersibility of
graphene and increase the conductivity of electrodes [31]. Choles-
terol was detected in a linear range between 50 mM and 10 mM
with a limit of detection of 1 mM. In addition, a 3-fold increase in the
current signal with the grapheneePVPePANi electrode was verified
when compared to an unmodified electrode, indicating the high
sensitivity of the biosensing system. The applicability of the paper-
based biosensor has also been tested by spiking different concen-
trations of cholesterol (0.05, 0.10, 0.25, 1.00, and 5.00 mM) into the
human serum, observing an acceptable linearity (r ¼ 0.9993) and
high sensor accuracy since the percentages of recoveries were
found in the range of 100e102% (RSD less than 5%).
Electrochemical sensors and biosensors have also been reported
to the detection of H2O2, which functions as a signalling molecule to
Table 1
Analytical parameters of sensors and biosensors with graphene.

Analyte detected Sensor type Electrode Sensing materials Linear range LODa Sensitivity (slope) Additional References
material information

Clinical applications
Glucose Electrochemical sensor GCE Grapheneecopper NP 0.4.5 mM 0.5 mM (S/N ¼ 3) 1234 mA mM
1 cm
2 r ¼ 0.9958 [25]

Cholesterol Electrochemical biosensor Graphite Graphene 50e300 mM 5 mM 443.25 mA mM
1 cm
2 r ¼ 0.9972 [28]
Electrochemical biosensor GCE Platinumepalladium 2.2e520 mM 0.75 mM (S/N ¼ 3) r ¼ 0.998 [29]
echitosanegraphene
hybrid nanocomposite
Optical biosensor Cerium oxideegraphene 12 mM to 7.2 mM 4.0 mM (S/N ¼ 3) r ¼ 0.993 [30]
composite

1
2
Electrochemical biosensor SPCE GrapheneePVPePANi 50 mM to 10 mM 1 mM (3y0 þ s) 34.77 mA mM cm r ¼ 0.9993 [31]

H2O2 Electrochemical sensor GCE Palladium NPegraphene 0.1e1000 mM 0.05 mM (S/N ¼ 3) [32]
nanosheet film
Electrochemical biosensor Graphite Grapheneepalladium 25e3500 mM 0.05 mM 92.82 mA mM
1 cm
2 r ¼ 0.998 [33]
oxide ehorseradish peroxidase
0.3 mM to 1.8 mM 0.11 mM (S/N ¼ 3) 2774.8 mA mM
1 cm
2

C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66

Electrochemical biosensor GCE Gold NPegraphene r ¼ 0.998 [26]
nanosheet film

DNA Magnetic field (Hall effect) GrapheneePMMA 1  10
3 to 10 nM 10 pM [23]

10
Dopamine Electrochemical sensor GCE Graphene and PVP 5  10 to 0.2 nM (S/N ¼ 3) RSD ¼ 2.2%; n ¼ 5 [34]
1  10
3 M r2 ¼ 0.999
Electrochemical sensor GCE Amino-group 0.2e38 mM 0.126 mM (S/N ¼ 3) [35]
functionalized Fe3O4
nanoparticles on graphene
sheets
Electrochemical sensor GCE Graphene 5.00e710 mM 2.00 mM 0.1125 mA mM
1 cm
2 r2 ¼ 0.9964 [36]

Ascorbic acid Electrochemical sensor GCE Amino-group 5e1600 mM 0.074 mM (S/N ¼ 3) [35]
functionalized Fe3O4
nanoparticles on graphene
sheets
Electrochemical sensor GCE Graphene 9.00e2314 mM 6.45 mM 0.06674 mA mM
1 cm
2 r2 ¼ 0.9979 [36]

Uric acid Electrochemical sensor GCE Amino-group 1e850 mM 0.056 mM (S/N ¼ 3) [35]
functionalized Fe3O4
nanoparticles on graphene
sheets
Electrochemical sensor GCE Graphene 6e1330 mM 4.82 mM r2 ¼ 0.9992 [36]

1
1 2
CEA Electrochemical immunosensor GCE Grapheneegold NP 0.010e50 ng mL 2.8 pg mL (S/ r ¼ 0.9847 [37]
ecarboxyl groups N ¼ 3)

1
Electrochemical immunosensor Grapheneegold NPesilver 0.010e120 ng mL 8 pg mL
1 (S/N ¼ 3) [38]
NP

a-fetoprotein Electrochemical immunosensor Grapheneegold NP 0.016e50 ng mL
1 5.4 pg mL
1 (S/ r2 ¼ 0.9892 [37]
ecarboxyl groups N ¼ 3)

1
Electrochemical immunosensor Doped graphene sheets 0.05e30 ng mL 5 pg mL
1 (S/N ¼ 3) 2
r ¼ 0.999 [39]

Thrombin Optical biosensor GCE Aptamer and graphene 1e100 fM 0.45 fM (S/N ¼ 3) r2 ¼ 0.9963 [40]
nanocomposite
Optical aptasensor Graphene 62.5e187.5 pM 31.3 pM r ¼ 0.993 [41]

PSA Electrochemical immunosensor GCE Grapheneemethylene blue/ 0.05e5.0 ng mL
1 13 pg mL
1 (S/ RSD ¼ 5.0%; n ¼ 5 [42]
chitosan nanocomposite N ¼ 3)
Electrochemical immunosensor GCE Grapheneesilver 0.01e10 ng mL
1 2 pg mL
1 3.6807 mA ng
1 mL
2 RSD ¼ 6.4%; n ¼ 5 [43]
hybridized mesoporous r2 ¼ 0.9963
silica NP

(continued on next page)

55
Table 1 (continued )

56
Analyte detected Sensor type Electrode Sensing materials Linear range LODa Sensitivity (slope) Additional References
material information

Electrochemical biosensor SPE Graphene nanosheets 2 pg mL
1 to 0.46 pg mL
1 (S/ RSD ¼ 6.43%; n ¼ 6 [44]
ehorseradish peroxidase 2 mg mL
1 N ¼ 3) r2 ¼ 0.9996
functionalized with gold NP

IL-6 Electrochemical immunosensor ITO Grapheneegold NP 1e40 pg mL
1 0.3 pg mL
1 [45]

1
1
2
MMP-2 Electrochemical immunosensor GCE Gold NPenitrogen doped 0.5 0.11 pg mL (S/ 4.20 mA ng mL RSD ¼ 5.7%; n ¼ 5 [46]
graphene composites e50,000 pg mL
1 N ¼ 3) r ¼ 0.997

cTnI Electrochemical immunosensor GCE Platinum NP/graphene 0.01e10 ng mL
1 0.0042 ng mL
1 (S/ RSD ¼ 4e11%; n ¼ 3 [24]
composite N ¼ 3) r ¼ 0.991

Environmental applications
Malachite green Electrochemical sensor GCE Graphene quantum dots 4.0  10
7 to 1.0  10
7 mol L
1 r ¼ 0.992 [47]
egold NP 1.0  10
5 mol L
1 (S/N ¼ 3)

Pb2þ Electrochemical sensor SPE Grapheneepoly(sodium 4- 0.5e120 mg L
1 0.089 mg L
1 [48]

C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66

styrenesulfonate)
composite
Electrochemical sensor GCE Grapheneepoly(crystal 2.00  10
8 to 1.0  10
8 mol L
1 [49]
violet) 1.95  10
5 mol L
1
Electrochemical sensor SPCE GrapheneeNafion 0.1e100 ng L
1 0.08 ng L
1 [50]

1
Cd 2þ
Electrochemical sensor SPE Grapheneepoly(sodium 4- 0.5e120 mg L 0.042 mg L
1 [48]
styrenesulfonate)
composite
Electrochemical sensor GCE Grapheneepoly(crystal 9.00  10
8 to 1.0  10
8 mol L
1 [49]
violet) 5.58  10
5 mol L
1
Electrochemical sensor SPCE GrapheneeNafion 0.1e100 ng L
1 0.06 ng L
1 [50]

13
8
Methyl parathion Electrochemical biosensor GCE Carboxylic grapheneeNiO 10 to 10 M 5  10
14 M [51]
Chlorpyrifos NPeNafion
Carbofuran 10
12 to 10
8 M 5  10
13 M

12
6
Paraoxon Photoelectrochemical sensor ITO GrapheneeMIPeCdSeeZnS 10 to 10 M 6.0  10
14 M r2 ¼ 0.975 [52]
Dichlorvos quantum dots 2.5  10
12 M r2 ¼ 0.993

4-aminophenol Photoelectrochemical sensor FTO GrapheneeMIPeCdS 5.0  10
8 to 2.3  10
8 M (S/ r ¼ 0.999 [53]
quantum dots 3.5  10
6 M N ¼ 3)

Hydrogen Electrochemical sensor Grapheneepalladium NP 20e1000 ppm 20 ppm [54]

Magnetic field (Hall effect) Grapheneepalladium NP 25e1000 ppm [55]

Acetylene Optical sensor Graphene 119.8 ppb r2 ¼ 0.9960 [56]

1
1
Hexachlorobenzene Electrochemical sensor GCE Nitrogen-doped graphene 3 mg L to 1.72 mg L (S/ RSD ¼ 3.9% (n ¼ 6) [57]
echitosan 10 mg L
1 N ¼ 3) r ¼ 0.993

Food applications
Erythromycin Electrochemical sensor Gold electrode GrapheneeMIPegold NP 7.0  10
8 to 2.3  10
8 M (S/ RSD ¼ 3.8% (n ¼ 5) [58]
echitosaneplatinum NP 9.0  10
5 M N ¼ 3) r ¼ 0.998

Tryptamine Electrochemical sensor GrapheneeMIPeMWCNT 9.0  10
8 to 7.4  10
8 M (S/ RSD ¼ 4.8% (n ¼ 3) [59]
7.0  10
5 M N ¼ 3) r ¼ 0.992

Staphylococcus aureus Piezoelectric sensor Gold electrode Grapheneeaptamer 4.1  101 to 41 cfu mL
1 r ¼ 0.9879 [60]
4.1  105 cfu mL
1 (3y0 þ s)

CEA: carcinoembryonic antigen; cTnI: cardiac troponin I; FTO: fluorine doped tin oxide; GCE: glassy carbon electrode; IL-6: interleukin-6; ITO: indium tin oxide electrode; LOD: limit of detection; MIP: molecularly imprinted
polymer; MMP-2: matrix metalloproteinase-2; MWCNT: multi-walled carbon nanotubes; NP: nanoparticles; PANi: polyaniline; PMMA: poly(methylmethacrylate); PVP: polyvinylpyrrolidone; RSD: residual standard deviation;
SPCE: screen-printed carbon electrode; SPE: screen-printed electrode.
a
Determination of LOD: “S/N ¼ 3”: LOD is three times the signal-to-noise ratio; “3y0 þ s”: LOD is 3 times the blank response (y0) plus the standard deviation (s).
 C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66
Fig. 1. (A) Electrogenerated chemiluminescence responses of luminol (0.15 mM) at cholesterol oxidaseecerium oxideeGCE (a), cholesterol oxidaseegrapheneeGCE (b), and
cholesterol oxidaseecerium oxide-grapheneeGCE (c) to 0.85 mM cholesterol. (Reprinted from Zhang et al. [30], Copyright (2013), with permission from Elsevier). (B) Amperometric
responses recorded at the GCE (curve a, blue); gold nanoparticles/GCE (curve b, red); graphene nanosheets/GCE (curve c, green) and graphene nanosheets/gold nanoparticles/GCE
(curve d, mauve) (Reprinted from Song et al. [26], Copyright (2013), with permission from Elsevier).
regulate various biological processes such as immune cell activities
[32]. For example, an amperometric biosensor was fabricated
combining gold nanoparticles and graphene nanosheets [26]
(Table 1). The biosensor displayed a linear response for the reduc-
tion of H2O2 in the range of 0.3e1.8 mM with a limit of detection of
0.11 mM, high sensitivity (2774.8 mA mM
1 cm
2), and a rapid
response, which reached 95% of the steady state condition within
5 s [26]. In addition, the successive introduction of graphene
nanosheets improves the analytical signal of biosensors, as shown
in Fig. 1B, which could be due to the catalytic effect of graphene
nanosheets. Other works reported respectively the fabrication of a
sensor based on palladium nanoparticles/graphene nanosheet film
coated on a GCE [32] and a biosensor based on the co-
immobilization of horseradish peroxidase and palladium on gra-
phene modified graphite electrode [33] to detect H2O2. Same limits
of detection were obtained in the two works (0.05 mM) but with
different linear ranges of response, that is, 0.1e1000 mM [32] and
25e3500 mM [33]. Good working and storage stability values were
also respectively verified, that is, when the working stability was
examined using the same sensor for ten repetitive measurements
in successive additions of 0.05 mM H2O2, a RSD of 3.2% was ob-
tained [32] and when the storage stability of the biosensor was
examined for a period of four weeks, the biosensor retained its
activity throughout the stability test [33]. This can be attributed to
the biocompatibility and stability of functionalized graphene and
palladium along with horseradish peroxidase, preventing the
enzyme discharge from the electrode.
Dopamine, ascorbic acid, and uric acid are important molecules
for physiological processes in the human metabolism; in the ma-
jority of works reporting the fabrication of biosensors, their
simultaneous detection is observed due to its importance in diag-
nostic research. In addition, dopamine, ascorbic acid, and uric acid
have similar electrochemical properties only differing in their
oxidation peak potentials at the electrode surface [35]. The fabri-
cation of electrochemical sensing platforms for the simultaneous
detection of ascorbic acid, dopamine, and uric acid has been
recently reported and based on functionalized Fe3O4 nanoparticles
on graphene sheets (limits of detection of 0.05 mM for dopamine,
0.15 mM for ascorbic acid, and 0.39 mM for uric acid) [35] and on
graphene (2.00 mM for dopamine, 6.45 mM for ascorbic acid, and
4.82 mM for uric acid) [36]. The lower limit of detection could be due
to the excellent electrocatalytic activity and biocompatibility of the
functionalized Fe3O4 nanoparticles/graphene sheets towards the-
ses biomolecules. For dopamine, another sensor based on graphene
sheets and PVP displayed the lowest limit of detection (0.2 nM) and
57
the largest linear range (5  10
10 to 1  10
3 M) [34] (Table 1). The
high selectivity towards dopamine from grapheneePVP sensor
could be explained by the strong adsorption property of PVP to
phenolic compounds in dopamine. The firm combination and
adsorption properties of PVP on the electrode surface could be the
cause to the good reproducibility (RSD of 2.16%) and stability (peak
currents remained more than 90.1% of their initial values when the
electrode was kept at 4 C for 10 days) [34].
Electrochemical immunosensors based on graphene sheets
were also fabricated to the detection of CEA and a-fetoprotein
[37e39], which are glycoproteins; CEA is considered the most used
tumour biomarker for the diagnosis of lung cancer, ovarian carci-
noma, and breast cancer and a-fetoprotein is widely used as a liver
cancer tumour marker [61]. For a-fetoprotein, electrochemical
immunosensors based on doped graphene sheets were fabricated
[37,39] with similar limit of detection (5.4 and 5 pg mL
1, respec-
tively). The doped graphene sheets can increase the electron
transfer and sorption due to their high conductivity, thus causing
sensitive analytical response and consequent satisfied limit of
detection.
Thrombin is a specific serine protease, having an important role
in various pathological conditions since it is produced in blood and
tissues when physiological and pathological blood coagulation
occur [62]. In Table 1, two optical biosensors are reported to its
detection, and based on a nanocomposite of aptamer and graphene
[40] and on a FRET aptasensor [41]. The optical biosensor based on
the combination of a nanocomposite of aptamer and graphene
displayed improved analytical performance with good selectivity, a
low detection limit of 0.45 fM, high sensitivity and with a linear
response up to 100 fM, associated to the role of graphene, which
greatly speeds up the charge transfer of the biosensor [40].
The diagnosis of the prostate cancer is commonly provided by
the determination of blood levels of the tumour marker PSA and
sensitive biosensors can be useful for the rapid and early screening
of prostate cancer. As reported in Table 1, a label-free immuno-
sensor based on graphene sheets and methylene blue/chitosan
nanocomposite was proposed to detect PSA [42], obtaining good
analytical performance (linear range of 0.05e5.0 ng mL
1 and
limit of detection of 13 pg mL
1). The large specific area of gra-
phene sheets, which could be used to adsorb a number of meth-
ylene blue molecules, the high electroactivity due to the
nanocomposite graphene sheetemethylene blue immobilized on
GCE, and the role of the chitosan, which functions as the disper-
sant, having a good biological function and compatibility, explain
the low limit of detection of the immunosensor. In addition, the
58 C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66
stability of the graphene sheetsemethylene blue film-modified
GCE immunosensor was examined by storage at 4 C, and no
obvious change was observed after 14-day storage, which in-
dicates that a good graphene sheetsemethylene blue film was very
efficient for retaining the bioactivity and preventing the bio-
molecules from leaking out because of the strong interaction be-
tween the electrode and these biomolecules [42]. A better limit of
detection (2 pg mL
1) was recently obtained [43] in an electro-
chemical immunosensor based on amino-functionalized graphene
with ferroceneecarboxaldehyde composite and silver hybridized
mesoporous silica nanoparticles were reported for the detection of
PSA. The good analytical performance can be due to the high
conductivity, good electron transfer property, and biocompati-
bility of the amino-functionalized graphene, which combined with
the ferroceneecarboxaldehyde composite on the high electrode
surface area, could amplified the analytical signal. The stability of
the immunosensor was also studied checking the current re-
sponses periodically. After one week, the current response of the
immunosensor decreased by 3.7%, and after three weeks, there
was no obvious signal change (RSD < 10%), meaning that the sta-
bility of the immunosensor was also acceptable [43]. The good
stability of the immunosensor may be due to the good stability,
large surface area, and large surface free energy of silver hybrid-
ized mesoporous silica nanoparticles, which ensure the stability of
antibody on the electrode surface, as well as its good biological
compatibility. A different approach to PSA detection was attemp-
ted by the development of a disposable electrochemical biosensor
based on graphene nanosheets and horseradish peroxidase func-
tionalized with gold nanoparticles immobilized on a carbon SPE,
which was prepared on sheets of vegetable parchments [44].
Graphene nanosheets were used to increase the conductivity and
the stability of the immunosensor due to their fast electron
transportation and good biocompatibility, and a good analytical
performance was obtained with the best limit of detection
(0.46 pg mL
1) and wider linear range (2 pg mL
1 to 2 mg mL
1)
over 6 orders of magnitude when compared with the other bio-
sensors reported in Table 1 for PSA detection. Different amounts of
PSA were also added into human serum sample for recovery tests,
and acceptable results with relative errors less than 7.6% and re-
coveries between 98.7 and 107.0% for the PSA recovery experiment
have been obtained, thus indicating good accuracy and precision
of the proposed method.
For the detection of MMP-2, which is an endopeptidase
expressed in the tumour growth invasion and metastasis, an elec-
trochemical immunosensor based on gold nanoparticles and
nitrogen-doped graphene composite [46] was reported, as shown
in Table 1. The nitrogen-doped graphene is more commonly used in
comparison to pristine graphene due to its much larger functional
surface area, higher ratio of surface active groups to volume, more
biocompatible CeN microenvironment, higher electrical conduc-
tivity, and more chemically active sites for further functionalization
[46]. A good limit of detection (0.11 pg mL
1) was obtained, which
could be attributed to the various advantages of the composite
constituted by gold nanoparticles and nitrogen-doped graphene
sheet suitable for biosensing, that is, facilitating robust immobili-
zation of antibodies, promoting electron transfer, and exhibiting
excellent electrochemical activity, thus enhancing the analytical
performance of the immunosensor due to the homogeneous
dispersion of gold nanoparticles on nitrogen graphene sheet [46].
The cTnI is a cardiac marker classified as a troponin, which is a
sensitive biomarker of myocardial cell injury [61]. An electro-
chemical immunosensor based on impedance prepared with
functionalized platinum nanoparticles immobilized on a graphene
monolayer was developed [24], where a limit of detection of
0.0042 ng mL
1 was obtained with reproducible results (RSD of
4e11%) and a linear range between 0.01 and 10 ng mL
1. The
combination of graphene monolayer composite with functionalized
platinum nanoparticles results in an increased biomolecular
immobilization for immunosensor fabrication, since graphene is an
ideal transducer material due to its large surface area, high het-
erogeneous electron transfer rates, high intrinsic mobility, and
good electrical and thermal conductivity, and the functionalized
platinum nanoparticles have a large specific surface area, excellent
biocompatibility, strong adsorption ability, and good conductivity
[24].
Heavy metal ions such as Pb2þ and Cd2þ were also detected by
graphene-based sensors [48e50]. For example, a disposable gra-
phene sensor based on SPE was fabricated to determine Cd2þ and
Pb2þ obtaining improved sensitivity and linearity (0.5e120 mg L
1),
which could be associated to the functionalization of graphene with
negatively charged poly(sodium 4-styrenesulfonate), which pro-
vides more absorbing sites [48].
Pesticides are also detected by graphene biosensors; an elec-
trochemical biosensor was fabricated through immobilization of
enzyme acetylcholinesterase on NiO nanoparticlesecarboxylic
grapheneeNafion modified GCE for the detection of methyl para-
thion, chlorpyrifos, and carbofuran [51] (Table 1). The biosensor
retained 91% of its initial electrochemical response, which indicates
its acceptable stability after a 30-day storage period. When applied
to real samples such as tap water and lake water samples, the re-
coveries observed by the biosensor were in the range of
93.0e105.2% [51].
4-Aminophenol was also detected by a graphene based sensor
with the presence of CdS quantum dots and molecularly imprinted
polymers (MIP) [53]. The photoelectrochemical sensor allowed the
rapid, sensitive, and selective detection of 4-aminophenol with a
low limit of detection (2.3  10
8 M) and excellent reproducibility
(RSD of 2.7%). The photoelectrochemical activity of quantum dots
can be enhanced through its hybridization with graphene due to
the improvement of the charge-separation rate of quantum dots,
which is associated to the excellent conductivity of graphene.
When applied to lake water samples, the recoveries obtained with
the sensor are in the range of 97.3 and 104.4%, indicating its feasi-
bility to determine 4-aminophenol in real water samples [53].
For food applications, graphene based sensors have been reported
to the detection of erythromycin [58] and tryptamine [59] through
electrochemical sensors, and to the detection of S. aureus through a
piezoelectric sensor [60]. The electrochemical sensor reported to the
detection of erythromycin was based on MIP on gold electrode
decorated by a nanocomposite constituted by chitosaneplatinum
nanoparticles and grapheneegold nanoparticles [58]. With the
introduction of grapheneegold nanoparticles, higher current
response was observed due to the synergistic effects of the good
electrical conductivity of graphene and gold nanoparticles [58]. A
low limit of detection (2.3  10
8 M) was achieved, which is better
than that obtained with a non-graphene based electrochemical
biosensor using acetylene black nanoparticles (8.0  10
8 M) [63].
When erythromycin analogs (kanamycin sulphate, neomycin sul-
phate, and spiramycin) were introduced into the biosensor, the
current response to erythromycin was much higher than that of the
other three interfering species, which indicates the high selectivity of
the biosensor to erythromycin [58]. After spiking milk and honey
samples, the recoveries of this sensor ranged from 95.8 to 103.0%,
which indicated the ability of the fabricated sensor to detect eryth-
romycin in real samples [58]. An electrochemical sensor based on
MIP, sulfonated graphene, and hyaluronic acideMWCNT composite
was proposed [59] for determination of tryptamine in cheese and
lactobacillus beverage samples. When the sensor surface was
modified with the sulfonated graphene layer, an increment of the
redox peak current was observed, which may be due to the high
 C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66
conductivity of graphene sheets. The recoveries obtained were in the
range of 91.5 and 103%, indicating that the sensor may be applied to
detection of tryptamine in real samples. In addition, the sensor has a
long-term stability, since it retained 87% of current response after
two weeks of storage and it could be regenerated with PBS. Recently,
a piezoelectric sensor was constructed with conjugation of graphene
and aptamers for the rapid and specific detection of S. aureus in milk
[60]. The incorporation of graphene allows the markedly change of
electric characteristics of the sensor electrode due to the good elec-
tronic conductivity of graphene, thus leading to a sensitive sensor
response. The constructed sensor demonstrated a linear relationship
between resonance frequency shifts with bacterial concentrations
ranging from 101 to 105 cfu mL
1 with a limit of detection of
41 cfu mL
1. The sensor could be regenerated, washing the electrode
with acetonitrile and acetone, and after re-bounding the aptamers to
graphene, the frequency shift of the S. aureus detection (81 Hz) was
very close to that of the initial sensor (83 Hz); after ten regenerations,
the frequency shift decreased by less than 5%, which indicates that
the sensor can be reused at least ten times [60].
2.2. Graphene oxide (GO)
Sensors and biosensors with GO have been used in: a) clinical
applications, for example, to the detection of glucose, DNA,
thrombin, and MMP-2; and, b) environmental applications, for
example, to the detection of Hg2þ, ammonia, and formaldehyde.
The introduction of GO [62,64] or the combination of GO with
nanoparticles [65] can also enhance the analytical response of
sensors and biosensors, mainly improving their sensitivity [65] and
limit of detection [64].
Table 2 shows the analytical parameters of sensors and bio-
sensors fabricated with GO and the analytical performance of some
examples listed in Table 2 is also discussed and compared in this
subsection.
The combination of platinum nanoparticles and GO was re-
ported in an electrochemical biosensor for glucose detection [65],
where the electrocatalytic activity is enhanced 12 times (charac-
terized in terms of amperometric sensitivity towards H2O2) when
compared to biosensors without GO. The effective surface area of
the biosensor based on the hybrid nanocomposite of GO and plat-
inum nanoparticles is significantly higher than for a platinum
biosensor, with an increase in electron transfer. When applied to
glucose detection, the sensitivity with platinum/GO biosensor
increased 25 times when compared to the platinum biosensor.
The sensitive and selective detection of DNA is very important
both in the clinical diagnosis and in the gene expression analysis
and the rapid and cost-effective biosensors are suggested as po-
tential analytical devices. For example, a chemiluminescence
biosensor for DNA detection was fabricated using GO and a
horseradish peroxidase-mimicking DNAzymes [68], which are
catalytic nucleic acids, commonly used as amplifying labels in
sensing platforms. GO was used in this optical biosensor due to its
high quenching efficiency, which could lead to the improvement of
the chemiluminescence emission intensity of the sensor, and a
good limit of detection (34 pM) was obtained. Another optical
biosensor (fluorescence) has incorporated an isothermal circular
strand-displacement polymerization reaction as a promising
amplification technique in the sensitive DNA detection, which lead
to a better limit of detection (4 pM) [64]. The GO acts as an efficient
signal to background ratio enhancer in the sensing platform,
defined as the ratio of fluorescence peak intensity from the target
(10 nM) to that from the blank [64]. In the absence of GO, this ratio
was 4.879 increasing to 17.83 upon addition of GO, demonstrating
that the proposed fluorescent DNA biosensor could be used for
sensitive DNA detection [64].
59
For the detection of thrombin, an optical aptasensor based on
FRET mechanism using multilayer GO [69] and an electrochemical
aptasensor [70] displayed limits of detection of 0.001 nM and
0.07 nM, respectively. The advantage of the multilayer GO FRET
sensor is the possibility of regeneration up to four times simply by
cleaning it with distilled water [69]. Another optical FRET biosensor
was reported for MMP-2 detection [72], where a limit of detection
of 2500 pg mL
1 was obtained (Table 2). The fluorescence
quenching capacity of GO and its unique adsorption characteristics
for biomolecules were advantageous to the fabrication of improved
sensors in terms of accuracy (RSD  7.03%) and rapid response (less
than 3 h) when applied to complex human serum samples. An
interesting work proposed the fabrication of a label-free impedi-
metric aptasensor to the detection of thrombin [62], and where the
biosensing performance of a wide set of graphenes prepared by
different methods was compared. Graphite oxide, GO, thermally
rGO, and electrochemically rGO were used in screen-printed elec-
trodes and Fig. 2 shows the impedance spectra recorded for the
various chemically modified graphenes together to the way of
preparation for each of them.
According to Fig. 2, the greatest change in impedimetric signal
after incubation with thrombin is from GO-based sensor due to its
most sensitive surface for thrombin detection in comparison to the
other three chemically modified graphenes.
As environmental applications, GO based sensors were pro-
posed to the detection of Hg2þ [73,74]. Electrochemical sensors
have been fabricated, obtaining limits of detection with different
orders of magnitude; with a gold-nanostructured sensor based on
GO [74], a limit of detection of 1.9 mg L
1 (1.9 ppb) was obtained,
which is lower the current WHO guidelines (6.0 ppb) [84], and with
a simple and sensitive electrochemical sensor based on functional
GO-ionic liquid composites with gold nanoparticles [73], a limit of
detection of 0.03 nM (0.006 ppb) was obtained in a wide linear
range (0.1e100 nM). In addition, such sensor can be regenerated by
immersion in a stirred solution of HNO3, KCl and EDTA, with the
possibility of its reuse with RSD of 2.6% [73]. Recently, a fluores-
cence biosensor (FRET principle) based on carbon dots-labelled
oligodeoxyribonucleotide and GO was proposed for Hg2þ detec-
tion [75], obtaining a higher limit of detection (2.6 nM) than that
obtained with the electrochemical sensor reported previously [73]
but also lower than the guideline values for drinking water. GO is
considered an effective oligonucleotide-based fluorescent sensing
platform material for Hg2þ detection with low background since it
could adsorb dye-labelled oligonucleotides via hydrophobic and
pep stacking interactions and simultaneously quench the fluo-
rescence of dyes by FRET principle. The feasibility of the sensor by
determining Hg2þ in citrus leaf samples was also evaluated
employing the standard addition method and agreements were
achieved between the results obtained by the reference method
and the sensor [75].
Table 2 also displays two works reporting the fabrication of
piezoelectric sensors for the detection of ammonia via quartz
crystal microbalance (QCM) [76,77]. For example, an ammonia
sensor with GO was reported, where the quality factor of the
piezoelectric sensor was analysed by its measurement with and
without the GO layer. It has been verified that with GO layer, the
quality factor is about two times larger than the same sensor
without GO in the whole detection range (100e1700 ppm). The
quality factor is ascribed to the high elasticity modulus of GO
layer, which produced the suppression of the surface energy loss
of the QCM and the use of GO as isolation layer provides a simple
way to realize high stability piezoelectric sensors based on QCM.
Recently, another QCM sensor was proposed for ammonia where
positively charged polyethylenimine and negatively charged GO
were assembled on the surfaces of cellulose acetate nanofibers
 60
Table 2
Analytical parameters of sensors and biosensors with graphene oxide (GO).

Analyte Sensor type Electrode Sensing materials Linear range LODa Additional information References
detected material

Glucose Electrochemical biosensor GOeplatinum NP 1 mM to 2 mM 1 mM r ¼ 0.999 [65]

Optical enzymatic GOesilver NPegold NP 2e5 mM 0.33 mM r2 ¼ 0.993 [66]
biosensor
Electrochemical sensor GCE GOegold NP 0.2e13.4 mM 0.05 mM [67]
2
DNA Optical biosensor GOehorseradish peroxidase- 0.1e3 nM 34 pM r ¼ 0.9914 [68]
mimicking DNAzymes

C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66

Optical biosensor GO 0.1e10 nM 4 pM [64]

Thrombin Electrochemical aptasensor GO 10e50 nM [62]

Optical biosensor GO 0.001 nM [69]
Electrochemical aptasensor Graphite GO 0.1e10 nM 0.07 nM (3y0 þ s) r ¼ 0.998 [70]
electrode

IL-6 Electrochemiluminescence GCE GO nanosheets/polyaniline 0.5e10,000 pg mL
1 0.17 pg mL
1 RSD ¼ 5.9%; n ¼ 3 [71]
immunosensor nanowires/CdSe quantum dots
nanocomposites

MMP-2 Optical biosensor GO 10,000e150,000 pg mL
1 2500 pg mL
1 RSD ¼ 0.53e4.56% [72]
2þ
Hg Electrochemical sensor GCE GOeionic liquid compositesegold 0.1e100 nM 0.03 nM (S/N ¼ 3) r ¼ 0.9808 [73]
NP
Electrochemical sensor SPCE GO 0.5e50 mg L
1 1.9 mg L
1 RSD ¼ 9.4% (n ¼ 8) r2 ¼ 0.9997 [74]
Optical sensor GOecarbon dots 5e200 nM 2.6 nM RSD ¼ 3.2% (n ¼ 11) r2 ¼ 0.974 [75]

Ammonia Piezoelectric sensor (QCM) GO 100e1700 ppm [76]

GOepolyethylenimine 1e50 ppm r2 ¼ 0.97 [77]

TNT Electrochemical sensor GO 0e20 ppm 6.74 ppm r2 ¼ 0.927 [78]

Optical sensor GOegold NPeaptamer 0.01e100 ng mL
1 3.6 pg mL
1 (S/N ¼ 3) RSD ¼ 1.62% (n ¼ 7) [79]
r ¼ 0.997
Optical sensor GOepeptides 4.40  10
9 mM to 4.40  10
12 mM [80]
4.40  10
4 mM

DNT Electrochemical sensor GO 0e20 ppm 2.73 ppm r2 ¼ 0.987 [78]

Formaldehyde Piezoelectric sensor (QCM) GO 0.5e3.5 ppm 0.06 ppm [81]

GO-amine functionalized silica NP 0.04 Hz mg
1 ppm
1 [82]

Dibutyl Electrochemical sensor GOeMIPeFe3O4egold NP 2.5  10
9 to 5.0  10
6 M 8.0  10
10 mol L
1 (S/ RSD ¼ 3.6% (n ¼ 6) [83]
phthalate N ¼ 3) r ¼ 0.998

CNT: carbon nanotubes; DNT: 2,4-dinitrotoluene; GCE: glassy carbon electrode; GO: graphene oxide; IL-6: interleukin-6; LOD: limit of detection; MMP-2: matrix metalloproteinase-2; NP: nanoparticles; QCM: quartz crystal
microbalance; RSD: residual standard deviation; SPCE: screen-printed carbon electrode; TNT: 2,4,6-trinitrotoluene.
a
Determination of LOD: “S/N ¼ 3”: LOD is three times the signal-to-noise ratio; “3y0 þ s”: LOD is 3 times the blank response (y0) plus the standard deviation (s).
 C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66 61

Fig. 2. Illustration for the preparation of chemically modified graphene materials with graphite as the starting material. (A) Graphite oxide was first generated via oxidation from
graphite. This was followed by: (B) thermal reduction/exfoliation of graphite oxide to yield thermally rGO; (C) exfoliation by ultrasonication to produce GO; and, (D) electrochemical
reduction of GO to generate electrochemically rGO, and respective impedance spectra (Nyquist plots,
Z00 vs. Z0 ) (Adapted from Loo et al. [62] with permission of The Royal Society of
Chemistry).

[77]. A good limit of detection (1 ppm) and high sensitivity (9 Hz)
were observed [77]. This result from the 3D structure of the
cellulose acetate nanofibers/polyethylenimine/GO composite and
well dispersion of GO on the sensor surface; the 3D structure also
provides high diffusivity channels for ammonia gas diffusion.
Recently, a QCM sensor using GO was fabricated for the detection
of formaldehyde [81] (Table 2). The good stability of such GO
QCM sensor (at least up to 100 days) could be due to the
reversibility of the adsorptionedesorption processes between the
functional groups of GO and formaldehyde, which occur due to
the break of their hydrogen bonding [81]. When the sensor was
exposed to a variety of common volatile chemicals such as
ethanol, acetone, chloroform, benzene, and toluene, the higher
response (frequency shift) was obtained to formaldehyde,
demonstrating its selectivity.
2.3. Reduced graphene oxide (rGO)
Sensors and biosensors with rGO have been used in: a) clinical
applications, for example, to the detection of glucose, H2O2, dopa-
mine, ascorbic acid, uric acid, and CEA; b) environmental applica-
tions, for example, to the detection of 17b-estradiol, bisphenol A,
hydrogen, and methane; and, c) food applications, for example, to
the detection of Pb2þ, nitrite, carbofuran, and carbaryl. The pres-
ence of rGO [85] or the combination of rGO with nanoparticles [86]
and polymers [87] can also enhance the analytical response of
sensors and biosensors, mainly improving their sensitivity.
Table 3 shows the analytical parameters of sensors and bio-
sensors fabricated with rGO and the analytical performance of
some examples listed in Table 3 is also discussed and compared in
this subsection.
For the detection of glucose, various electrochemical sensors
and biosensors with rGO have been reported in the latest years, as
shown in Table 3. For example, an electrochemical biosensor was
constructed with rGO, PPy, and gold nanoparticles [90]. The re-
ported high sensitivity (123.8 mA mM
1 cm
2) could be due to the
large surface area and the conductivity of rGO but also to the
electron transfer promotion from the gold nanoparticles homoge-
neously distributed onto the rGO surface. It can also be concluded
that within the biocompatible nanocomposite, the glucose oxidase
may well-contacted with gold nanoparticles, PPy, and rGO, which
facilitated the fast electron transfer with a relatively low barrier
between the enzyme and the electrode. In addition, the nano-
composite may be also promising in encapsulating some other
biomolecules, suggesting a prospective in fabricating versatile
electrochemical biosensors and other devices [90].
Non-enzymatic glucose sensors with rGO have been also
developed in recent years, obtaining good analytical performance
in terms of limit of detection and sensitivity. With such works, it is
also observed that the synergy effect between rGO and, for
example, nanoparticles can enhance the analytical response of
sensors, thus obtaining improved linear range and sensitivity. For
example, electrochemical sensors for glucose detection were based
on modified GCE with rGO and NiO nanofibers [91] or Co3O4
nanowires [92]. Limits of detection of 0.77 mM and 25 nM and
sensitivities of 1100 and 3390 mA mM
1 cm
2 were respectively
obtained. The electrocatalytic activity and large electrochemical
capacitance of Co3O4 nanowires as well as the large surface area
and high conductivity of graphene contribute to the good sensing
performance towards glucose detection. The good limit of detection
of NiO nanofibers based sensor is also attributed to the large
microscopic surface area and good catalytic activity of Ni nanofibers
and superior electrochemical properties of rGO. A more recent
work [93] reported the fabrication of a non-enzymatic glucose
electrochemical sensor based on rGO also combining platinum and
copper oxide nanoparticles, where an excellent sensitivity
Table 3

62
Analytical parameters of sensors and biosensors with reduced graphene oxide (rGO).

Analyte detected Sensor type Electrode Sensing materials Linear range LODa Sensitivity (slope) Additional information References
material

Clinical applications
Glucose Electrochemical enzymatic GCE rGOePAMAMesilver NP 0.032e1.89 mM 4.5 mM (S/N ¼ 3) 75.72 mA mM
1 cm
2 RSD ¼ 5.6%; n ¼ 6 [88]
biosensor r ¼ 0.996
Electrochemical enzymatic rGOeTiO2 NP 0.032e1.67 mM 4.8 mM (S/N ¼ 3) 35.8 mA mM
1 cm
2 r ¼ 0.997 [89]
biosensor
Electrochemical enzymatic rGO 0.1e27 mM 1.85 mA mM
1 cm
2 [85]
biosensor

1
2
Electrochemical enzymatic GCE rGOePPyegold NP 0.2e1.2 mM 123.8 mA mM cm RSD ¼ 4.1%; n ¼ 8 [90]
biosensor r ¼ 0.986
Electrochemical sensor GCE rGOeNiO nanofiberseNafion 2.0 mM to 0.6 mM 0.77 mM (S/N ¼ 3) 1100 mA mM
1 cm
2 r ¼ 0.9978 [91]
Electrochemical sensor GCE rGOeCo3O4 nanowires 0e80 mM 25 nM 3390 mA mM
1 cm
2 [92]
Electrochemical sensor SPE rGOeplatinum NPCuO NP 0e12 mM 0.01 mM (S/N ¼ 3) 3577 mA mM
1 cm
2 r ¼ 0.9991 [93]
Optical sensor rGO 1 nM to 10 mM 1 nM [94]

H2O2 Electrochemical biosensor rGOeMWCNTeplatinum NP 10 pM to 0.19 nM and 6 pM (3y0 þ s) 1.99 mA pM
1 cm
2 and RSD ¼ 3.3%; n ¼ 5 [95]

C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66

emyoglobin 0.25 nM to 2.24 mM 0.037 mA nM
1 cm
2

DNA Electrochemical biosensor GCE Gold NPerGOehorseradish 10
17 to 10
13 M 5 aM (S/N ¼ 3) [96]
peroxidase functionalized carbon
spheres

Dopamine Electrochemical biosensor GCE Hemin-functionalized rGO 0.5e40 mM 0.17 mM (S/N ¼ 3) r2 ¼ 0.9988 [97]
Ascorbic acid 1e100 mM 0.3 mM (S/N ¼ 3) r2 ¼ 0.9989
Uric acid 0.5e50 mM 0.17 mM (S/N ¼ 3) r2 ¼ 0.9948

CEA Electrochemical GCE rGOesilver NP 0.5 pg mL
1 to 0.12 pg mL
1 (S/ RSD ¼ 6.5% r ¼ 0.9994 [86]
immunosensor 0.5 ng mL
1 N ¼ 3)
Electrochemical paper rGO 2e8 ng mL
1 25.8 mA ng
1 mL cm
2 RSD ¼ 7.6% (n ¼ 5) [87]
sensor r2 ¼ 0.998

Environmental applications
17b-estradiol Electrochemical sensor rGOeMIPeFe3O4 nanoparticles 0.05e10 mM 0.819 nM [98]
4-nitrophenol Electrochemical sensor GCE rGOeMIP 0.01e100 mM 0.005 mM (S/N ¼ 3) RSD ¼ 4.4% (n ¼ 8) [99]
r ¼ 0.9991
Bisphenol A Electrochemical sensor GCE rGOemagnetic nanoparticles 6.0  10
8 to 1.7  10
8 mol L
1 RSD ¼ 2.69% (n ¼ 5) [100]
echitosan 1.1  10
5 mol L
1 (S/N ¼ 3) r ¼ 0.9977

Methane gas Optical sensor rGOeCNTePMMA 10e100 ppm [101]

Nitrogen dioxide Piezoelectric sensor (QCM) rGOePVP 20e100 ppm [102]

rGOeSnO2esilver NP 2.05e20.5 mg cm
3 [103]

Organophosphorous Piezoelectric sensor (QCM) rGO 0e50 mg m3 [104]

neurotoxin

Food applications
Pb2þ Electrochemical sensor SPCE rGO 5e200 ppb 1 ppb (S/N ¼ 3) r2 ¼ 0.9923 [105]

Nitrite Electrochemical sensor GCE rGOeFe3O4 nanoparticles 1.0  10
6 to 3  10
7 M (S/ [106]
9.2  10
5 M N ¼ 3)

Carbofuran Electrochemical sensor GCE rGOeCoO nanoparticles 0.2e70 mM 4.2 mg L
1 (S/N ¼ 3) r ¼ 0.9996 [107]
Carbaryl 0.5e200 mM 7.5 mg L
1 (S/N ¼ 3) r ¼ 0.9995

CEA: carcinoembryonic antigen; CNT: carbon nanotubes; GCE: glassy carbon electrode; LOD: limit of detection; MIP: molecularly imprinted polymer; NP: nanoparticles; PAMAM: polyamidoamine dendrimer; PMMA: pol-
y(methylmethacrylate); PPy: polypyrrole; PVP: polyvinylpyrrolidone; rGO: reduced graphene oxide; QCM: quartz crystal microbalance; RSD: residual standard deviation; SPCE: screen-printed carbon electrode; SPE: screen-
printed electrode;
a
Determination of LOD: “S/N ¼ 3”: LOD is three times the signal-to-noise ratio; “3y0 þ s”: LOD is 3 times the blank response (y0) plus the standard deviation (s).
 C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66
(3577 mA mM
1 cm
2) and very good limit of detection (0.01 mM)
were obtained, as shown in Table 3. In addition, the analytical
response in various sensors based on electrode (SPE), SPEeplati-
numerGO, SPEecopper oxideerGO, as well as SPEeplati-
numecopper oxideerGO, was studied, as shown in Fig. 3, where
the voltammograms obtained for the different sensors are observed
when applied to glucose detection. The anodic peak is well defined
on SPEeplatinumecopper oxide-rGO (curve e), which means that
copper oxide is responsible for electrocatalytic oxidation of glucose
and platinum is necessary to increase the sensitivity.
As shown in Fig. 3, the anodic peak is well defined on
PteCuOerGOeSPE (curve e), which means that CuO is responsible
for electrocatalytic oxidation of glucose and platinum is necessary
to increase the sensitivity [69]. In addition, an improved shelf
storage stability of the sensors since after preserving the sensors in
air tight container for three months, a decrease in current response
less than 2% was found [93]. Recently, an optical sensor based on
etched fibre Bragg gratings coated with functionalized rGO was
used for glucose detection [94], obtaining an excellent limit of
detection of 1 nM with a linear range from 1 nM to 10 mM. The
specificity of the sensor to glucose molecules was confirmed from
the observation of a very small change in the shift in Bragg wave-
length (~4 pM) even for 10 mM lactose solution. The literature also
provided the comparison of the analytical signal between two
graphene related materials. Electrochemical enzymatic biosensors
were fabricated with GO and rGO for glucose detection and it has
been verified that rGO based biosensor displays a higher current
signal when compared to a GO based biosensor, which indicates
that the rGO promotes a fast electron transfer process between the
enzyme and the graphene material [85].
H2O2 biosensors based on the electrocatalysis of myoglobin on
rGOemulti-walled CNT (MWCNT)eplatinum nanoparticles com-
posite were proposed [95], achieving a limit of detection of 6 pM.
The combination of the properties of 3D hierarchical rGOeMWCNT
hybrid, the good electrocatalytic property of platinum nano-
particles, and the presence of a large amount of electroactive
myoglobin explain such low limit of detection [95].
Another interesting work reported for the first time the fabri-
cation of an electrochemical DNA biosensor based on labelled gold
nanoparticles and rGO with horseradish peroxidase [96]. Such tri-
ple nanostructure based on rGO could be the cause for the limit of
detection in the attomolar range (5 aM) due to the high conduc-
tivity of gold nanoparticleseDNA and the signal amplification from
a signal tag of horseradish peroxidase functionalized carbon sphere
Fig. 3. Voltammograms of SPE (a), activated SPE with H2SO4 (b); PterGOeSPE (c);
CuOerGOeSPE (d); and, PteCuOerGOeSPE (e) in 0.1 M NaOH containing 3 mM
glucose (Reprinted from Dhara et al. [93], Copyright (2014), with permission from
Elsevier).
63
that relates each target hybridization event to multiple enzyme
reaction [96].
For the simultaneous detection of ascorbic acid, dopamine, and
uric acid, an electrochemical biosensor was recently proposed
based on hemin-functionalized rGO [97], obtaining a good analyt-
ical performance (Table 3). The large surface area of rGO can be
responsible to the electrochemical biosensor response, that is, to its
high electrocatalytic activity and excellent electrical conductivity;
rGO possesses a rich surface chemistry property and has the po-
tential to further promote the catalytic activity and stability of
hemin through cationep or pep stacking. In addition, the hemin-
functionalized rGO has enhanced electrostatic interaction and good
conductivity due to the remarkable synergistic effects of hemin and
graphene, and a large number of carboxylic functional groups
attached on the surface of hemin and functionalized rGO could
provide a selective interface via hydrogen bonds, thus leading to an
improved biosensor [97].
For CEA detection, an electrochemical immunosensor was con-
structed using rGO and silver nanoparticles [86]. Through linear
sweep stripping voltammetric curves, it was verified that the curves
obtained for rGO modified electrode showed a sharp stripping peak
in relation to the curves obtained for electrode without GO, which
indicates that the graphene efficiently improved the detection
sensitivity due to its ability to accelerate the electron transfer be-
tween deposited silver nanoparticles and the electrode. In addition, it
was also verified that after the electrochemical reduction of GO to
obtain rGO, the resulting immunosensor showed further enhanced
stripping peak, leading to higher sensitivity [86]. Recently, a paper
based sensor was fabricated with rGO and a conducting polymer
(poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate),
PEDOT:PSS) to the detection of CEA [87]. After the incorporation of
rGO into the conducting paper, both electrochemical performance
and signal stability were improved. That is, the conductivity of
PEDOT:PSS paper with rGO (3.12  10
2 S cm
1) has increased 270-
fold when compared with the conductivity of PEDOT:PSS paper
(1.16  10
4 S cm
1), which demonstrates the excellent electro-
chemical activity and faster charge transfer kinetics of the rGO/
PEDOT:PSS conducting paper film [87]. Thus, the developed paper
electrode is a promising alternative over the expensive conventional
electrodes such as ITO, gold and glassy carbon, and able to be used as
flexible and environment friendly platform for cancer biomarker
detection with high sensitivity (25.8 mA ng
1 mL cm
2) [87].
The detection of 17b-estradiol in water was performed through
an electrochemical sensor based on rGO, MIP, and magnetic
nanoparticles (Fe3O4 nanobeads) [98]. A low limit of detection
(0.819 nM) was obtained, which could result from the amplification
of the response current caused by the Fe3O4 nanobeads/graphene
complex [98]. Bisphenol A was also detected by an electrochemical
sensor based on rGO, chitosan, and magnetic nanoparticles, where
rGO acts as an effective electron promoter for electrocatalytic
oxidation of bisphenol A and the electrode constituted by the
composite of magnetic nanoparticles and rGO can lower the
oxidation overpotential of bisphenol A, improving the sensitivity
for its determination [100]. A good analytical performance was
verified with a limit of detection of 1.7  10
8 mol L
1 and RSD of
2.69%, which indicated an excellent reproducibility; after two
weeks, 92.3% of the initial response was retained, indicating good
sensor stability [100].
For the detection of hydrogen, an optical gas sensor based on
SPR using rGO and gold nanoparticles was used in order to combine
the semiconducting and photocatalytic activity behaviour of the
rGO with the SPR of gold nanoparticles for the simultaneous
detection of hydrogen and nitrogen dioxide [108].
In the area of food research, sensors and biosensors with rGO
were applied, for example, to the detection of Pb2þ, nitrite,
64 C.I.L. Justino et al. / Trends in Analytical Chemistry 91 (2017) 53e66
carbofuran, and carbaryl, as reported in Table 3. Heavy metals such
as Pb2þ were detected in foods sources since this contamination is a
very serious problem in developing countries. Disposable sensors
were constructed based on rGO for the determination of Pb2þ in
real tap water, juices, preserved eggs, and tea samples [105]. The
sensors displayed improved electrochemical properties after the
deposition of rGO onto the electrode since it increases the active
sites of the electrode surface, promoting higher electron transfer
[105]. Although the sensor displayed a wide linear response
(5e200 ppb) for detection of Pb2þ with high sensitivity, minimal
interferences of Cd2þ (<50 ppb) and Cu2þ (<100 ppb) were verified.
Nitrite was also detected by a sensitive electrochemical sensor
based on rGO and Fe3O4 magnetic nanoparticles [106]. A large value
of heterogeneous electron transfer constant was obtained, which
indicated the high ability of rGO as electron carrier for the pro-
motion of electron transfer between the Fe3O4 nanoparticles and
the electrode surface. The sensor was applied to real samples of
sausage in order to demonstrate the good performance of the
sensor and it was found that there is a good agreement between the
values obtained by the sensor and the reference method, and also
between the spiked and found values [106]. Carbofuran and
carbaryl were also detected by graphene sensors using rGO and
CoO nanoparticles [107]. The catalytic performance of CoO was
improved due to the combination of rGO, which contributes to the
enhancement of the charge transfer efficiency. Fruits and vegeta-
bles (grapes, oranges, tomato, and cabbages) were used as real
samples in order to evaluate the practical application of the fabri-
cated electrochemical sensor. The satisfactory recoveries
(96e104%) confirmed the stable and sensitive sensor for real
sample analysis, and a good accuracy was also reported since the
analytical results of the sensor were in agreement with those ob-
tained with a reference method (HPLC) [107].
3. Conclusions and future perspectives
This review reports recent works on the application of graphene
and graphene related materials on sensors and biosensors in clin-
ical analysis, environmental monitoring, or food quality control.
The analytical performance of various sensing systems was
compared and the role of such materials on the improvement of the
analytical response and thus performance of sensors and bio-
sensors was also discussed.
The graphene and graphene related materials are mainly used in
sensors and biosensors following electrochemical principles due to
their large accessible surface area, electrical conductivity, high
electron transfer rate provided, and capacity to immobilize
enzymes.
The most recent progress in this field corresponds to the syn-
thesis of nanocomposites combining graphene or graphene related
materials with other compounds such as metal nanoparticles,
allowing the fabrication of electrochemical sensors and biosensors
with improved analytical performance since the combination of
electrical characteristics of nanocomposite constituents make ad-
vantageous its integration on such sensing platforms. Comparing
with the publications before 2012, more papers reported the
fabrication of sensors and biosensors combining graphene and MIP,
thus synthesising different composites. According to Justino et al.
[109], MIP belong to the group of recent recognition elements used
in sensors and biosensors, and they aroused the attention of re-
searchers due to their high selectivity, stability, short time of syn-
thesis, high thermostability, and cost effectiveness. However, the
lack of surface functionality of graphene and low solubility in water
and most organic solvents could limit its practical application in
biosensors. Thus, great attention was paid to GO (the oxygenated
derivative of graphene), as one of graphene related material, due to
its remarkable properties such as good water dispersibility and
biocompatibility, large surface area, facile surface modification and
low manufacturing cost.
Future interesting research trends in the field of the present
paper will include the improvement of portable sensing platforms
based on graphene and graphene related materials, as well as
simultaneous multiplexed detection of analytes, as already
occurred for CNT biosensors [1]. The fabrication of graphene sen-
sors and biosensors as point-of care devices, which could lead to
better in situ clinical diagnosis or as in situ sensing platforms for
environmental analysis should also be performed in future
research. In addition, the combination of graphene with MIP should
be more studied in order to improve the analytical performance of
applied sensors and biosensors, taking advantage of the direct
contact of grapheneeMIP and the analyte. The fabrication of other
composites based on graphene and elements such as nanoparticles
and organic polymers have been identified as an improvement of
recent graphene based sensors and biosensors, and the research
should be directed accordingly in order to improve their analytical
performance, mainly in terms of reproducibility and limit of
detection, as well as real-time analysis.
Acknowledgements
This work was funded by Portuguese Science Foundation (FCT)
through scholarships (ref. SFRH/BPD/95961/2013) under POCH
funds, co-financed by the European Social Fund and Portuguese
National Funds from MEC. This work was also funded by national
funds through FCT/MEC (PIDDAC) under project IF/00407/2013/
CP1162/CT0023. Thanks are also due, for the financial support to
CESAM (UID/AMB/50017), to FCT/MEC through national funds, and
the co-funding by the FEDER, within the PT2020 Partnership
Agreement and Compete 2020.
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