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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, Laboratorio 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 strength (200 times greater than steel), high electrical conductivity,
which is 60-fold greater than single-walled CNT (SWCNT), and six
In the latest years, the construction of sensors and biosensors orders of magnitude higher than copper, and high elasticity and
has been improved with the incorporation of various nanomaterials thermal conductivity [5e7].
such as nanowires synthesized from metals (e.g., Ni, Cu, Au, Pt), Other related materials, which belong to the graphene family,
metal oxides (ZnO, SnO2, Fe2O3), and silicon/indium/gallium are the graphene oxide (GO) and the reduced GO (rGO). GO is
semiconductors (Si, InP, GaN), quantum dots based on CdSe, CdTe or layered and oxygenated graphene sheets that contain oxygen
CdSeTe, carbon nanotubes (CNT), and metal nanoparticles (based functional groups, such as epoxides, carboxyls, hydroxyls, and al-
on Au, Cu, Pd, Co, Ag, or Pt) due to their conductive properties, high cohols, located on the edge and surface of the sheet with a carbon
surface-to-volume ratio, and good biocompatibility, thus contrib- to oxygen ratio of approximately 3:1 [8]. The presence of oxygen
uting to the improvement of the analytical performance of such functional groups on the graphene surface may enhance the het-
sensors and biosensors [1e3]. Recently, graphene has dominated erogeneous electron and charge transfer rate and make GO water-
the scientific interest in order to compete with such nanomaterials soluble and biocompatible [9]. After reduction, GO transforms into
to the fabrication of sensors and biosensors. rGO with some residual oxygen and structural defects, yielding
Graphene is a planar sheet of carbon atoms and it is the basic high thermal conductivity comparable to that of doped conductive
structural element of graphite, CNT, and fullerenes. The carbon polymers, about 36 times higher than that of Si and about 100 times
bonds are sp2 hybridized, where the in-plane sCeC bond is one of higher than that of GaAs [10].
the strongest bonds in materials and the out-of-plane p bond is Due to its planar shape and chemical structure, the graphene
responsible for the electron conduction of graphene, providing the has various advantageous properties in comparison to other ma-
weak interaction among graphene layers or between graphene and terials to the construction of sensors and biosensors. For example,
substrate [4]. Such electron configuration is the basis to the large the atomic thickness of the graphene sheet and its extremely high
surface area of graphene (2630 m2 g1), which is higher than that of surface-to-volume ratio make such material highly sensitive to-
graphite (10 m2 g1) and CNT (1315 m2 g1), high mechanical 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-
* Corresponding author. Department of Chemistry & CESAM, University of Aveiro, tivity than silicon nanowires or CNT, thus contributing to the
Campus de Santiago, 3810-193 Aveiro, Portugal. Fax: þ351 232 910 183. fabrication of improved electrochemical sensors and biosensors
E-mail address: celinejustino@ua.pt (C.I.L. Justino).

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 as well as hydrogen; and, c) food applications, for example, to the
regarding its application in electrochemical sensors and biosensors, detection of erythromycin, tryptamine, and Staphylococcus aureus.
that is, graphene does not contain metallic impurities, which The combination of graphene sheets with polyvinylpyrrolidone
interfere with the material electrochemistry, and the production of (PVP) [23] or nanoparticles [24e26] can enhance the analytical
graphene used graphite, which is cheap and accessible [12]. In response of sensors and biosensors, mainly improving their sensi-
addition, graphene can be easily connected to biomolecules by pep tivity [24,27], limit of detection [27], and reproducibility [27].
stacking and hydrophobic interactions and the lower noise ratio, Table 1 shows the analytical parameters of sensors and biosensors
facile functionalization, solution-processibility, and biocompati- fabricated with graphene and the analytical performance of some
bility are advantages of graphene as channel material [9,11]. examples listed in Table 1 is also discussed and compared in this
The graphene functionalization can be achieved with: a) poly- subsection.
mers, from their in situ synthesis obtaining high-strength and An electrochemical sensor based on glassy carbon electrode
conductive materials; for example, the GO/polyaniline (PANi) (GCE) with copper nanoparticles and graphene was proposed to the
composites can be prepared by the in situ polymerization of aniline detection of glucose [25]. The combination of such nanomaterials
in GO dispersion; b) nanoparticles, which immobilization in gra- provided better analytical response than the same sensors without
phene occurs through hydrophobic and static interactions and graphene, that is, the electrochemical sensor also exhibited much
promotes high conductivity and excellent thermal stability [7]. The better electrocatalytic properties for glucose oxidation when
fabrication of graphene/nanoparticles composites is commonly compared to sensor only with copper nanoparticles, which is
based on in situ growth and solution mixing by reducing metal ions attributed to the large surface area, high conductivity, and fast
since the oxygenated groups on the surfaces of both GO and gra- electron transfer provided by graphene sheets.
phene facilitate the nucleation of metal nanoparticles; and, c) To the detection of cholesterol, a graphene electrochemical
biomolecules such as DNA, proteins, peptides, and cellulose, which enzymatic biosensor onto graphite electrodes [28] and an
can also be incorporated in graphene surface through physical electrochemical enzymatic biosensor based on a plati-
adsorption or chemical bonding, modifying its structure due to the numepalladiumechitosanegraphene hybrid nanocomposite onto
various surface functional groups of such biomolecules [7]. In the GCE [29] were fabricated. The analytical performance of the gra-
latest five years, various papers reviewed the graphene based phene hybrid nanocomposite biosensor (limit of detection of
sensors and biosensors in different perspectives, and directed to 0.75 mM and linear range between 2.2 and 520 mM) [29] was better
general sensors and biosensors [13e16], electrochemical sensors than the graphene biosensor (limit of detection of 5 mM and linear
and biosensors [5,6,11,12,17e19], and optical sensors [9,20], and for range between 50 and 300 mM) [28]. The presence of the hybrid
the detection of biomarkers [5], heavy metals [8], and glucose [21]. nanocomposite together with the unique catalytic activity and
In the present review, the literature concerning the construction chemical selectivity of the bimetallic nanoparticles not only could
of sensors and biosensors based on graphene and graphene related accelerate the direct electron transfer from the redox enzyme to the
materials are critically updated, covering the period of 2012e2016, electrode surface, but also could enhance the immobilized amount
for the detection of various analytes reported in clinical, environ- of cholesterol oxidase, thus enhancing its electrochemical proper-
mental, and food research areas. The analytical performance of ties and consequently its analytical performance for the detection
graphene based sensors and biosensors and their advantages and of cholesterol. The construction of low-cost, simple, and sensitive
limitations are compared, and some approaches are also reported optical enzymatic biosensors based on electrogenerated chem-
in order to improve their analytical performance. iluminescence principle, where cerium oxide and graphene com-
posite were immobilized onto GCE, was reported [30]. A
2. Graphene and graphene related materials based sensors comparative study has been done by using three kinds of bio-
and biosensors sensors based on cholesterol oxidaseecerium oxideeGCE, choles-
terol oxidaseegrapheneeGCE, and cholesterol oxidaseecerium
In the latest years, a large number of sensors and biosensors based oxideegrapheneeGCE, as shown in Fig. 1A. The electrogenerated
on graphene and graphene related materials (GO and rGO) have been chemiluminescence intensity of luminol has been enhanced to a
reported in various areas such as clinical, environmental, and food much greater extent in the presence of cerium oxideegraphene,
sciences research. Although the majority of sensors and biosensors which is attributed to its electrocatalytic properties since more
has an electrochemical transduction principle, optical and piezo- efficient active sites have been created for the catalytic oxidation of
electric sensors and biosensors have been also fabricated, as shown H2O2 to produce various reactive oxygen species that can enhance
in the following subsections. The analytical performance of sensors the chemiluminescence intensity of luminol.
and biosensors has been evaluated through various analytical figures Recently, a paper-based cholesterol biosensor was proposed
of merit such as sensitivity, limit of detection, repeatability, and using a novel nanocomposite of grapheneePVPePANi, verifying
reproducibility, which are commonly used to validate an analytical that the presence of small amount of PVP (2 mg mL1) in the
method, ensuring the quality of the results [22]. The transduction nanocomposites can substantially improve the dispersibility of
mechanism and the morphological characteristics of sensors can graphene and increase the conductivity of electrodes [31]. Choles-
influence their sensitivity and limit of detection and such figures of terol was detected in a linear range between 50 mM and 10 mM
merit should be evaluated in order to assess, respectively, the with a limit of detection of 1 mM. In addition, a 3-fold increase in the
analytical reliability and capacity of sensors [22]. current signal with the grapheneePVPePANi electrode was verified
when compared to an unmodified electrode, indicating the high
2.1. Graphene sensitivity of the biosensing system. The applicability of the paper-
based biosensor has also been tested by spiking different concen-
Sensors and biosensors with graphene have been used in: a) trations of cholesterol (0.05, 0.10, 0.25, 1.00, and 5.00 mM) into the
clinical applications, for example, to the detection of glucose, human serum, observing an acceptable linearity (r ¼ 0.9993) and
cholesterol, H2O2, dopamine, ascorbic acid, uric acid, CEA, a-feto- high sensor accuracy since the percentages of recoveries were
protein, thrombin, and PSA; b) environmental applications, for found in the range of 100e102% (RSD less than 5%).
example, to the detection of metal ions such as Cd2þ and Pb2þ, Electrochemical sensors and biosensors have also been reported
pesticides such as methyl parathion, chlorpyrifos, and carbofuran, 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 mM1 cm2 r ¼ 0.9958 [25]

Cholesterol Electrochemical biosensor Graphite Graphene 50e300 mM 5 mM 443.25 mA mM1 cm2 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 mM1 cm2 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 mM1 cm2

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  103 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  103 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 mM1 cm2 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 mM1 cm2 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 mL1 (S/N ¼ 3) [38]
NP

a-fetoprotein Electrochemical immunosensor Grapheneegold NP 0.016e50 ng mL1 5.4 pg mL1 (S/ r2 ¼ 0.9892 [37]
ecarboxyl groups N ¼ 3)
1
Electrochemical immunosensor Doped graphene sheets 0.05e30 ng mL 5 pg mL1 (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 mL1 13 pg mL1 (S/ RSD ¼ 5.0%; n ¼ 5 [42]
chitosan nanocomposite N ¼ 3)
Electrochemical immunosensor GCE Grapheneesilver 0.01e10 ng mL1 2 pg mL1 3.6807 mA ng1 mL2 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 mL1 to 0.46 pg mL1 (S/ RSD ¼ 6.43%; n ¼ 6 [44]
ehorseradish peroxidase 2 mg mL1 N ¼ 3) r2 ¼ 0.9996
functionalized with gold NP

IL-6 Electrochemical immunosensor ITO Grapheneegold NP 1e40 pg mL1 0.3 pg mL1 [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 mL1 N ¼ 3) r ¼ 0.997

cTnI Electrochemical immunosensor GCE Platinum NP/graphene 0.01e10 ng mL1 0.0042 ng mL1 (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  107 to 1.0  107 mol L1 r ¼ 0.992 [47]
egold NP 1.0  105 mol L1 (S/N ¼ 3)

Pb2þ Electrochemical sensor SPE Grapheneepoly(sodium 4- 0.5e120 mg L1 0.089 mg L1 [48]

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


styrenesulfonate)
composite
Electrochemical sensor GCE Grapheneepoly(crystal 2.00  108 to 1.0  108 mol L1 [49]
violet) 1.95  105 mol L1
Electrochemical sensor SPCE GrapheneeNafion 0.1e100 ng L1 0.08 ng L1 [50]
1
Cd 2þ
Electrochemical sensor SPE Grapheneepoly(sodium 4- 0.5e120 mg L 0.042 mg L1 [48]
styrenesulfonate)
composite
Electrochemical sensor GCE Grapheneepoly(crystal 9.00  108 to 1.0  108 mol L1 [49]
violet) 5.58  105 mol L1
Electrochemical sensor SPCE GrapheneeNafion 0.1e100 ng L1 0.06 ng L1 [50]
13 8
Methyl parathion Electrochemical biosensor GCE Carboxylic grapheneeNiO 10 to 10 M 5  1014 M [51]
Chlorpyrifos NPeNafion
Carbofuran 1012 to 108 M 5  1013 M
12 6
Paraoxon Photoelectrochemical sensor ITO GrapheneeMIPeCdSeeZnS 10 to 10 M 6.0  1014 M r2 ¼ 0.975 [52]
Dichlorvos quantum dots 2.5  1012 M r2 ¼ 0.993

4-aminophenol Photoelectrochemical sensor FTO GrapheneeMIPeCdS 5.0  108 to 2.3  108 M (S/ r ¼ 0.999 [53]
quantum dots 3.5  106 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 L1 N ¼ 3) r ¼ 0.993

Food applications
Erythromycin Electrochemical sensor Gold electrode GrapheneeMIPegold NP 7.0  108 to 2.3  108 M (S/ RSD ¼ 3.8% (n ¼ 5) [58]
echitosaneplatinum NP 9.0  105 M N ¼ 3) r ¼ 0.998

Tryptamine Electrochemical sensor GrapheneeMIPeMWCNT 9.0  108 to 7.4  108 M (S/ RSD ¼ 4.8% (n ¼ 3) [59]
7.0  105 M N ¼ 3) r ¼ 0.992

Staphylococcus aureus Piezoelectric sensor Gold electrode Grapheneeaptamer 4.1  101 to 41 cfu mL1 r ¼ 0.9879 [60]
4.1  105 cfu mL1 (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 57

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 the largest linear range (5  1010 to 1  103 M) [34] (Table 1). The
[32]. For example, an amperometric biosensor was fabricated high selectivity towards dopamine from grapheneePVP sensor
combining gold nanoparticles and graphene nanosheets [26] could be explained by the strong adsorption property of PVP to
(Table 1). The biosensor displayed a linear response for the reduc- phenolic compounds in dopamine. The firm combination and
tion of H2O2 in the range of 0.3e1.8 mM with a limit of detection of adsorption properties of PVP on the electrode surface could be the
0.11 mM, high sensitivity (2774.8 mA mM1 cm2), and a rapid cause to the good reproducibility (RSD of 2.16%) and stability (peak
response, which reached 95% of the steady state condition within currents remained more than 90.1% of their initial values when the
5 s [26]. In addition, the successive introduction of graphene electrode was kept at 4 C for 10 days) [34].
nanosheets improves the analytical signal of biosensors, as shown Electrochemical immunosensors based on graphene sheets
in Fig. 1B, which could be due to the catalytic effect of graphene were also fabricated to the detection of CEA and a-fetoprotein
nanosheets. Other works reported respectively the fabrication of a [37e39], which are glycoproteins; CEA is considered the most used
sensor based on palladium nanoparticles/graphene nanosheet film tumour biomarker for the diagnosis of lung cancer, ovarian carci-
coated on a GCE [32] and a biosensor based on the co- noma, and breast cancer and a-fetoprotein is widely used as a liver
immobilization of horseradish peroxidase and palladium on gra- cancer tumour marker [61]. For a-fetoprotein, electrochemical
phene modified graphite electrode [33] to detect H2O2. Same limits immunosensors based on doped graphene sheets were fabricated
of detection were obtained in the two works (0.05 mM) but with [37,39] with similar limit of detection (5.4 and 5 pg mL1, respec-
different linear ranges of response, that is, 0.1e1000 mM [32] and tively). The doped graphene sheets can increase the electron
25e3500 mM [33]. Good working and storage stability values were transfer and sorption due to their high conductivity, thus causing
also respectively verified, that is, when the working stability was sensitive analytical response and consequent satisfied limit of
examined using the same sensor for ten repetitive measurements detection.
in successive additions of 0.05 mM H2O2, a RSD of 3.2% was ob- Thrombin is a specific serine protease, having an important role
tained [32] and when the storage stability of the biosensor was in various pathological conditions since it is produced in blood and
examined for a period of four weeks, the biosensor retained its tissues when physiological and pathological blood coagulation
activity throughout the stability test [33]. This can be attributed to occur [62]. In Table 1, two optical biosensors are reported to its
the biocompatibility and stability of functionalized graphene and detection, and based on a nanocomposite of aptamer and graphene
palladium along with horseradish peroxidase, preventing the [40] and on a FRET aptasensor [41]. The optical biosensor based on
enzyme discharge from the electrode. the combination of a nanocomposite of aptamer and graphene
Dopamine, ascorbic acid, and uric acid are important molecules displayed improved analytical performance with good selectivity, a
for physiological processes in the human metabolism; in the ma- low detection limit of 0.45 fM, high sensitivity and with a linear
jority of works reporting the fabrication of biosensors, their response up to 100 fM, associated to the role of graphene, which
simultaneous detection is observed due to its importance in diag- greatly speeds up the charge transfer of the biosensor [40].
nostic research. In addition, dopamine, ascorbic acid, and uric acid The diagnosis of the prostate cancer is commonly provided by
have similar electrochemical properties only differing in their the determination of blood levels of the tumour marker PSA and
oxidation peak potentials at the electrode surface [35]. The fabri- sensitive biosensors can be useful for the rapid and early screening
cation of electrochemical sensing platforms for the simultaneous of prostate cancer. As reported in Table 1, a label-free immuno-
detection of ascorbic acid, dopamine, and uric acid has been sensor based on graphene sheets and methylene blue/chitosan
recently reported and based on functionalized Fe3O4 nanoparticles nanocomposite was proposed to detect PSA [42], obtaining good
on graphene sheets (limits of detection of 0.05 mM for dopamine, analytical performance (linear range of 0.05e5.0 ng mL1 and
0.15 mM for ascorbic acid, and 0.39 mM for uric acid) [35] and on limit of detection of 13 pg mL1). The large specific area of gra-
graphene (2.00 mM for dopamine, 6.45 mM for ascorbic acid, and phene sheets, which could be used to adsorb a number of meth-
4.82 mM for uric acid) [36]. The lower limit of detection could be due ylene blue molecules, the high electroactivity due to the
to the excellent electrocatalytic activity and biocompatibility of the nanocomposite graphene sheetemethylene blue immobilized on
functionalized Fe3O4 nanoparticles/graphene sheets towards the- GCE, and the role of the chitosan, which functions as the disper-
ses biomolecules. For dopamine, another sensor based on graphene sant, having a good biological function and compatibility, explain
sheets and PVP displayed the lowest limit of detection (0.2 nM) and 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 4e11%) and a linear range between 0.01 and 10 ng mL1. The
GCE immunosensor was examined by storage at 4 C, and no combination of graphene monolayer composite with functionalized
obvious change was observed after 14-day storage, which in- platinum nanoparticles results in an increased biomolecular
dicates that a good graphene sheetsemethylene blue film was very immobilization for immunosensor fabrication, since graphene is an
efficient for retaining the bioactivity and preventing the bio- ideal transducer material due to its large surface area, high het-
molecules from leaking out because of the strong interaction be- erogeneous electron transfer rates, high intrinsic mobility, and
tween the electrode and these biomolecules [42]. A better limit of good electrical and thermal conductivity, and the functionalized
detection (2 pg mL1) was recently obtained [43] in an electro- platinum nanoparticles have a large specific surface area, excellent
chemical immunosensor based on amino-functionalized graphene biocompatibility, strong adsorption ability, and good conductivity
with ferroceneecarboxaldehyde composite and silver hybridized [24].
mesoporous silica nanoparticles were reported for the detection of Heavy metal ions such as Pb2þ and Cd2þ were also detected by
PSA. The good analytical performance can be due to the high graphene-based sensors [48e50]. For example, a disposable gra-
conductivity, good electron transfer property, and biocompati- phene sensor based on SPE was fabricated to determine Cd2þ and
bility of the amino-functionalized graphene, which combined with Pb2þ obtaining improved sensitivity and linearity (0.5e120 mg L1),
the ferroceneecarboxaldehyde composite on the high electrode which could be associated to the functionalization of graphene with
surface area, could amplified the analytical signal. The stability of negatively charged poly(sodium 4-styrenesulfonate), which pro-
the immunosensor was also studied checking the current re- vides more absorbing sites [48].
sponses periodically. After one week, the current response of the Pesticides are also detected by graphene biosensors; an elec-
immunosensor decreased by 3.7%, and after three weeks, there trochemical biosensor was fabricated through immobilization of
was no obvious signal change (RSD < 10%), meaning that the sta- enzyme acetylcholinesterase on NiO nanoparticlesecarboxylic
bility of the immunosensor was also acceptable [43]. The good grapheneeNafion modified GCE for the detection of methyl para-
stability of the immunosensor may be due to the good stability, thion, chlorpyrifos, and carbofuran [51] (Table 1). The biosensor
large surface area, and large surface free energy of silver hybrid- retained 91% of its initial electrochemical response, which indicates
ized mesoporous silica nanoparticles, which ensure the stability of its acceptable stability after a 30-day storage period. When applied
antibody on the electrode surface, as well as its good biological to real samples such as tap water and lake water samples, the re-
compatibility. A different approach to PSA detection was attemp- coveries observed by the biosensor were in the range of
ted by the development of a disposable electrochemical biosensor 93.0e105.2% [51].
based on graphene nanosheets and horseradish peroxidase func- 4-Aminophenol was also detected by a graphene based sensor
tionalized with gold nanoparticles immobilized on a carbon SPE, with the presence of CdS quantum dots and molecularly imprinted
which was prepared on sheets of vegetable parchments [44]. polymers (MIP) [53]. The photoelectrochemical sensor allowed the
Graphene nanosheets were used to increase the conductivity and rapid, sensitive, and selective detection of 4-aminophenol with a
the stability of the immunosensor due to their fast electron low limit of detection (2.3  108 M) and excellent reproducibility
transportation and good biocompatibility, and a good analytical (RSD of 2.7%). The photoelectrochemical activity of quantum dots
performance was obtained with the best limit of detection can be enhanced through its hybridization with graphene due to
(0.46 pg mL1) and wider linear range (2 pg mL1 to 2 mg mL1) the improvement of the charge-separation rate of quantum dots,
over 6 orders of magnitude when compared with the other bio- which is associated to the excellent conductivity of graphene.
sensors reported in Table 1 for PSA detection. Different amounts of When applied to lake water samples, the recoveries obtained with
PSA were also added into human serum sample for recovery tests, the sensor are in the range of 97.3 and 104.4%, indicating its feasi-
and acceptable results with relative errors less than 7.6% and re- bility to determine 4-aminophenol in real water samples [53].
coveries between 98.7 and 107.0% for the PSA recovery experiment For food applications, graphene based sensors have been reported
have been obtained, thus indicating good accuracy and precision to the detection of erythromycin [58] and tryptamine [59] through
of the proposed method. electrochemical sensors, and to the detection of S. aureus through a
For the detection of MMP-2, which is an endopeptidase piezoelectric sensor [60]. The electrochemical sensor reported to the
expressed in the tumour growth invasion and metastasis, an elec- detection of erythromycin was based on MIP on gold electrode
trochemical immunosensor based on gold nanoparticles and decorated by a nanocomposite constituted by chitosaneplatinum
nitrogen-doped graphene composite [46] was reported, as shown nanoparticles and grapheneegold nanoparticles [58]. With the
in Table 1. The nitrogen-doped graphene is more commonly used in introduction of grapheneegold nanoparticles, higher current
comparison to pristine graphene due to its much larger functional response was observed due to the synergistic effects of the good
surface area, higher ratio of surface active groups to volume, more electrical conductivity of graphene and gold nanoparticles [58]. A
biocompatible CeN microenvironment, higher electrical conduc- low limit of detection (2.3  108 M) was achieved, which is better
tivity, and more chemically active sites for further functionalization than that obtained with a non-graphene based electrochemical
[46]. A good limit of detection (0.11 pg mL1) was obtained, which biosensor using acetylene black nanoparticles (8.0  108 M) [63].
could be attributed to the various advantages of the composite When erythromycin analogs (kanamycin sulphate, neomycin sul-
constituted by gold nanoparticles and nitrogen-doped graphene phate, and spiramycin) were introduced into the biosensor, the
sheet suitable for biosensing, that is, facilitating robust immobili- current response to erythromycin was much higher than that of the
zation of antibodies, promoting electron transfer, and exhibiting other three interfering species, which indicates the high selectivity of
excellent electrochemical activity, thus enhancing the analytical the biosensor to erythromycin [58]. After spiking milk and honey
performance of the immunosensor due to the homogeneous samples, the recoveries of this sensor ranged from 95.8 to 103.0%,
dispersion of gold nanoparticles on nitrogen graphene sheet [46]. which indicated the ability of the fabricated sensor to detect eryth-
The cTnI is a cardiac marker classified as a troponin, which is a romycin in real samples [58]. An electrochemical sensor based on
sensitive biomarker of myocardial cell injury [61]. An electro- MIP, sulfonated graphene, and hyaluronic acideMWCNT composite
chemical immunosensor based on impedance prepared with was proposed [59] for determination of tryptamine in cheese and
functionalized platinum nanoparticles immobilized on a graphene lactobacillus beverage samples. When the sensor surface was
monolayer was developed [24], where a limit of detection of modified with the sulfonated graphene layer, an increment of the
0.0042 ng mL1 was obtained with reproducible results (RSD of 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 59

conductivity of graphene sheets. The recoveries obtained were in the For the detection of thrombin, an optical aptasensor based on
range of 91.5 and 103%, indicating that the sensor may be applied to FRET mechanism using multilayer GO [69] and an electrochemical
detection of tryptamine in real samples. In addition, the sensor has a aptasensor [70] displayed limits of detection of 0.001 nM and
long-term stability, since it retained 87% of current response after 0.07 nM, respectively. The advantage of the multilayer GO FRET
two weeks of storage and it could be regenerated with PBS. Recently, sensor is the possibility of regeneration up to four times simply by
a piezoelectric sensor was constructed with conjugation of graphene cleaning it with distilled water [69]. Another optical FRET biosensor
and aptamers for the rapid and specific detection of S. aureus in milk was reported for MMP-2 detection [72], where a limit of detection
[60]. The incorporation of graphene allows the markedly change of of 2500 pg mL1 was obtained (Table 2). The fluorescence
electric characteristics of the sensor electrode due to the good elec- quenching capacity of GO and its unique adsorption characteristics
tronic conductivity of graphene, thus leading to a sensitive sensor for biomolecules were advantageous to the fabrication of improved
response. The constructed sensor demonstrated a linear relationship sensors in terms of accuracy (RSD  7.03%) and rapid response (less
between resonance frequency shifts with bacterial concentrations than 3 h) when applied to complex human serum samples. An
ranging from 101 to 105 cfu mL1 with a limit of detection of interesting work proposed the fabrication of a label-free impedi-
41 cfu mL1. The sensor could be regenerated, washing the electrode metric aptasensor to the detection of thrombin [62], and where the
with acetonitrile and acetone, and after re-bounding the aptamers to biosensing performance of a wide set of graphenes prepared by
graphene, the frequency shift of the S. aureus detection (81 Hz) was different methods was compared. Graphite oxide, GO, thermally
very close to that of the initial sensor (83 Hz); after ten regenerations, rGO, and electrochemically rGO were used in screen-printed elec-
the frequency shift decreased by less than 5%, which indicates that trodes and Fig. 2 shows the impedance spectra recorded for the
the sensor can be reused at least ten times [60]. various chemically modified graphenes together to the way of
preparation for each of them.
2.2. Graphene oxide (GO) According to Fig. 2, the greatest change in impedimetric signal
after incubation with thrombin is from GO-based sensor due to its
Sensors and biosensors with GO have been used in: a) clinical most sensitive surface for thrombin detection in comparison to the
applications, for example, to the detection of glucose, DNA, other three chemically modified graphenes.
thrombin, and MMP-2; and, b) environmental applications, for As environmental applications, GO based sensors were pro-
example, to the detection of Hg2þ, ammonia, and formaldehyde. posed to the detection of Hg2þ [73,74]. Electrochemical sensors
The introduction of GO [62,64] or the combination of GO with have been fabricated, obtaining limits of detection with different
nanoparticles [65] can also enhance the analytical response of orders of magnitude; with a gold-nanostructured sensor based on
sensors and biosensors, mainly improving their sensitivity [65] and GO [74], a limit of detection of 1.9 mg L1 (1.9 ppb) was obtained,
limit of detection [64]. which is lower the current WHO guidelines (6.0 ppb) [84], and with
Table 2 shows the analytical parameters of sensors and bio- a simple and sensitive electrochemical sensor based on functional
sensors fabricated with GO and the analytical performance of some GO-ionic liquid composites with gold nanoparticles [73], a limit of
examples listed in Table 2 is also discussed and compared in this detection of 0.03 nM (0.006 ppb) was obtained in a wide linear
subsection. range (0.1e100 nM). In addition, such sensor can be regenerated by
The combination of platinum nanoparticles and GO was re- immersion in a stirred solution of HNO3, KCl and EDTA, with the
ported in an electrochemical biosensor for glucose detection [65], possibility of its reuse with RSD of 2.6% [73]. Recently, a fluores-
where the electrocatalytic activity is enhanced 12 times (charac- cence biosensor (FRET principle) based on carbon dots-labelled
terized in terms of amperometric sensitivity towards H2O2) when oligodeoxyribonucleotide and GO was proposed for Hg2þ detec-
compared to biosensors without GO. The effective surface area of tion [75], obtaining a higher limit of detection (2.6 nM) than that
the biosensor based on the hybrid nanocomposite of GO and plat- obtained with the electrochemical sensor reported previously [73]
inum nanoparticles is significantly higher than for a platinum but also lower than the guideline values for drinking water. GO is
biosensor, with an increase in electron transfer. When applied to considered an effective oligonucleotide-based fluorescent sensing
glucose detection, the sensitivity with platinum/GO biosensor platform material for Hg2þ detection with low background since it
increased 25 times when compared to the platinum biosensor. could adsorb dye-labelled oligonucleotides via hydrophobic and
The sensitive and selective detection of DNA is very important pep stacking interactions and simultaneously quench the fluo-
both in the clinical diagnosis and in the gene expression analysis rescence of dyes by FRET principle. The feasibility of the sensor by
and the rapid and cost-effective biosensors are suggested as po- determining Hg2þ in citrus leaf samples was also evaluated
tential analytical devices. For example, a chemiluminescence employing the standard addition method and agreements were
biosensor for DNA detection was fabricated using GO and a achieved between the results obtained by the reference method
horseradish peroxidase-mimicking DNAzymes [68], which are and the sensor [75].
catalytic nucleic acids, commonly used as amplifying labels in Table 2 also displays two works reporting the fabrication of
sensing platforms. GO was used in this optical biosensor due to its piezoelectric sensors for the detection of ammonia via quartz
high quenching efficiency, which could lead to the improvement of crystal microbalance (QCM) [76,77]. For example, an ammonia
the chemiluminescence emission intensity of the sensor, and a sensor with GO was reported, where the quality factor of the
good limit of detection (34 pM) was obtained. Another optical piezoelectric sensor was analysed by its measurement with and
biosensor (fluorescence) has incorporated an isothermal circular without the GO layer. It has been verified that with GO layer, the
strand-displacement polymerization reaction as a promising quality factor is about two times larger than the same sensor
amplification technique in the sensitive DNA detection, which lead without GO in the whole detection range (100e1700 ppm). The
to a better limit of detection (4 pM) [64]. The GO acts as an efficient quality factor is ascribed to the high elasticity modulus of GO
signal to background ratio enhancer in the sensing platform, layer, which produced the suppression of the surface energy loss
defined as the ratio of fluorescence peak intensity from the target of the QCM and the use of GO as isolation layer provides a simple
(10 nM) to that from the blank [64]. In the absence of GO, this ratio way to realize high stability piezoelectric sensors based on QCM.
was 4.879 increasing to 17.83 upon addition of GO, demonstrating Recently, another QCM sensor was proposed for ammonia where
that the proposed fluorescent DNA biosensor could be used for positively charged polyethylenimine and negatively charged GO
sensitive DNA detection [64]. 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 mL1 0.17 pg mL1 RSD ¼ 5.9%; n ¼ 3 [71]
immunosensor nanowires/CdSe quantum dots
nanocomposites

MMP-2 Optical biosensor GO 10,000e150,000 pg mL1 2500 pg mL1 RSD ¼ 0.53e4.56% [72]

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 L1 1.9 mg L1 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 mL1 3.6 pg mL1 (S/N ¼ 3) RSD ¼ 1.62% (n ¼ 7) [79]
r ¼ 0.997
Optical sensor GOepeptides 4.40  109 mM to 4.40  1012 mM [80]
4.40  104 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 mg1 ppm1 [82]

Dibutyl Electrochemical sensor GOeMIPeFe3O4egold NP 2.5  109 to 5.0  106 M 8.0  1010 mol L1 (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) shown in Table 3. For example, an electrochemical biosensor was
were observed [77]. This result from the 3D structure of the constructed with rGO, PPy, and gold nanoparticles [90]. The re-
cellulose acetate nanofibers/polyethylenimine/GO composite and ported high sensitivity (123.8 mA mM1 cm2) could be due to the
well dispersion of GO on the sensor surface; the 3D structure also large surface area and the conductivity of rGO but also to the
provides high diffusivity channels for ammonia gas diffusion. electron transfer promotion from the gold nanoparticles homoge-
Recently, a QCM sensor using GO was fabricated for the detection neously distributed onto the rGO surface. It can also be concluded
of formaldehyde [81] (Table 2). The good stability of such GO that within the biocompatible nanocomposite, the glucose oxidase
QCM sensor (at least up to 100 days) could be due to the may well-contacted with gold nanoparticles, PPy, and rGO, which
reversibility of the adsorptionedesorption processes between the facilitated the fast electron transfer with a relatively low barrier
functional groups of GO and formaldehyde, which occur due to between the enzyme and the electrode. In addition, the nano-
the break of their hydrogen bonding [81]. When the sensor was composite may be also promising in encapsulating some other
exposed to a variety of common volatile chemicals such as biomolecules, suggesting a prospective in fabricating versatile
ethanol, acetone, chloroform, benzene, and toluene, the higher electrochemical biosensors and other devices [90].
response (frequency shift) was obtained to formaldehyde, Non-enzymatic glucose sensors with rGO have been also
demonstrating its selectivity. developed in recent years, obtaining good analytical performance
in terms of limit of detection and sensitivity. With such works, it is
2.3. Reduced graphene oxide (rGO) also observed that the synergy effect between rGO and, for
example, nanoparticles can enhance the analytical response of
Sensors and biosensors with rGO have been used in: a) clinical sensors, thus obtaining improved linear range and sensitivity. For
applications, for example, to the detection of glucose, H2O2, dopa- example, electrochemical sensors for glucose detection were based
mine, ascorbic acid, uric acid, and CEA; b) environmental applica- on modified GCE with rGO and NiO nanofibers [91] or Co3O4
tions, for example, to the detection of 17b-estradiol, bisphenol A, nanowires [92]. Limits of detection of 0.77 mM and 25 nM and
hydrogen, and methane; and, c) food applications, for example, to sensitivities of 1100 and 3390 mA mM1 cm2 were respectively
the detection of Pb2þ, nitrite, carbofuran, and carbaryl. The pres- obtained. The electrocatalytic activity and large electrochemical
ence of rGO [85] or the combination of rGO with nanoparticles [86] capacitance of Co3O4 nanowires as well as the large surface area
and polymers [87] can also enhance the analytical response of and high conductivity of graphene contribute to the good sensing
sensors and biosensors, mainly improving their sensitivity. performance towards glucose detection. The good limit of detection
Table 3 shows the analytical parameters of sensors and bio- of NiO nanofibers based sensor is also attributed to the large
sensors fabricated with rGO and the analytical performance of microscopic surface area and good catalytic activity of Ni nanofibers
some examples listed in Table 3 is also discussed and compared in and superior electrochemical properties of rGO. A more recent
this subsection. work [93] reported the fabrication of a non-enzymatic glucose
For the detection of glucose, various electrochemical sensors electrochemical sensor based on rGO also combining platinum and
and biosensors with rGO have been reported in the latest years, as 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 mM1 cm2 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 mM1 cm2 r ¼ 0.997 [89]
biosensor
Electrochemical enzymatic rGO 0.1e27 mM 1.85 mA mM1 cm2 [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 mM1 cm2 r ¼ 0.9978 [91]
Electrochemical sensor GCE rGOeCo3O4 nanowires 0e80 mM 25 nM 3390 mA mM1 cm2 [92]
Electrochemical sensor SPE rGOeplatinum NPCuO NP 0e12 mM 0.01 mM (S/N ¼ 3) 3577 mA mM1 cm2 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 pM1 cm2 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 nM1 cm2

DNA Electrochemical biosensor GCE Gold NPerGOehorseradish 1017 to 1013 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 mL1 to 0.12 pg mL1 (S/ RSD ¼ 6.5% r ¼ 0.9994 [86]
immunosensor 0.5 ng mL1 N ¼ 3)
Electrochemical paper rGO 2e8 ng mL1 25.8 mA ng1 mL cm2 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  108 to 1.7  108 mol L1 RSD ¼ 2.69% (n ¼ 5) [100]
echitosan 1.1  105 mol L1 (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 cm3 [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  106 to 3  107 M (S/ [106]
9.2  105 M N ¼ 3)

Carbofuran Electrochemical sensor GCE rGOeCoO nanoparticles 0.2e70 mM 4.2 mg L1 (S/N ¼ 3) r ¼ 0.9996 [107]
Carbaryl 0.5e200 mM 7.5 mg L1 (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 63

(3577 mA mM1 cm2) and very good limit of detection (0.01 mM) that relates each target hybridization event to multiple enzyme
were obtained, as shown in Table 3. In addition, the analytical reaction [96].
response in various sensors based on electrode (SPE), SPEeplati- For the simultaneous detection of ascorbic acid, dopamine, and
numerGO, SPEecopper oxideerGO, as well as SPEeplati- uric acid, an electrochemical biosensor was recently proposed
numecopper oxideerGO, was studied, as shown in Fig. 3, where based on hemin-functionalized rGO [97], obtaining a good analyt-
the voltammograms obtained for the different sensors are observed ical performance (Table 3). The large surface area of rGO can be
when applied to glucose detection. The anodic peak is well defined responsible to the electrochemical biosensor response, that is, to its
on SPEeplatinumecopper oxide-rGO (curve e), which means that high electrocatalytic activity and excellent electrical conductivity;
copper oxide is responsible for electrocatalytic oxidation of glucose rGO possesses a rich surface chemistry property and has the po-
and platinum is necessary to increase the sensitivity. tential to further promote the catalytic activity and stability of
As shown in Fig. 3, the anodic peak is well defined on hemin through cationep or pep stacking. In addition, the hemin-
PteCuOerGOeSPE (curve e), which means that CuO is responsible functionalized rGO has enhanced electrostatic interaction and good
for electrocatalytic oxidation of glucose and platinum is necessary conductivity due to the remarkable synergistic effects of hemin and
to increase the sensitivity [69]. In addition, an improved shelf graphene, and a large number of carboxylic functional groups
storage stability of the sensors since after preserving the sensors in attached on the surface of hemin and functionalized rGO could
air tight container for three months, a decrease in current response provide a selective interface via hydrogen bonds, thus leading to an
less than 2% was found [93]. Recently, an optical sensor based on improved biosensor [97].
etched fibre Bragg gratings coated with functionalized rGO was For CEA detection, an electrochemical immunosensor was con-
used for glucose detection [94], obtaining an excellent limit of structed using rGO and silver nanoparticles [86]. Through linear
detection of 1 nM with a linear range from 1 nM to 10 mM. The sweep stripping voltammetric curves, it was verified that the curves
specificity of the sensor to glucose molecules was confirmed from obtained for rGO modified electrode showed a sharp stripping peak
the observation of a very small change in the shift in Bragg wave- in relation to the curves obtained for electrode without GO, which
length (~4 pM) even for 10 mM lactose solution. The literature also indicates that the graphene efficiently improved the detection
provided the comparison of the analytical signal between two sensitivity due to its ability to accelerate the electron transfer be-
graphene related materials. Electrochemical enzymatic biosensors tween deposited silver nanoparticles and the electrode. In addition, it
were fabricated with GO and rGO for glucose detection and it has was also verified that after the electrochemical reduction of GO to
been verified that rGO based biosensor displays a higher current obtain rGO, the resulting immunosensor showed further enhanced
signal when compared to a GO based biosensor, which indicates stripping peak, leading to higher sensitivity [86]. Recently, a paper
that the rGO promotes a fast electron transfer process between the based sensor was fabricated with rGO and a conducting polymer
enzyme and the graphene material [85]. (poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate),
H2O2 biosensors based on the electrocatalysis of myoglobin on PEDOT:PSS) to the detection of CEA [87]. After the incorporation of
rGOemulti-walled CNT (MWCNT)eplatinum nanoparticles com- rGO into the conducting paper, both electrochemical performance
posite were proposed [95], achieving a limit of detection of 6 pM. and signal stability were improved. That is, the conductivity of
The combination of the properties of 3D hierarchical rGOeMWCNT PEDOT:PSS paper with rGO (3.12  102 S cm1) has increased 270-
hybrid, the good electrocatalytic property of platinum nano- fold when compared with the conductivity of PEDOT:PSS paper
particles, and the presence of a large amount of electroactive (1.16  104 S cm1), which demonstrates the excellent electro-
myoglobin explain such low limit of detection [95]. chemical activity and faster charge transfer kinetics of the rGO/
Another interesting work reported for the first time the fabri- PEDOT:PSS conducting paper film [87]. Thus, the developed paper
cation of an electrochemical DNA biosensor based on labelled gold electrode is a promising alternative over the expensive conventional
nanoparticles and rGO with horseradish peroxidase [96]. Such tri- electrodes such as ITO, gold and glassy carbon, and able to be used as
ple nanostructure based on rGO could be the cause for the limit of flexible and environment friendly platform for cancer biomarker
detection in the attomolar range (5 aM) due to the high conduc- detection with high sensitivity (25.8 mA ng1 mL cm2) [87].
tivity of gold nanoparticleseDNA and the signal amplification from The detection of 17b-estradiol in water was performed through
a signal tag of horseradish peroxidase functionalized carbon sphere 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  108 mol L1 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
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
detection of hydrogen and nitrogen dioxide [108].
glucose (Reprinted from Dhara et al. [93], Copyright (2014), with permission from In the area of food research, sensors and biosensors with rGO
Elsevier). 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 its remarkable properties such as good water dispersibility and
as Pb2þ were detected in foods sources since this contamination is a biocompatibility, large surface area, facile surface modification and
very serious problem in developing countries. Disposable sensors low manufacturing cost.
were constructed based on rGO for the determination of Pb2þ in Future interesting research trends in the field of the present
real tap water, juices, preserved eggs, and tea samples [105]. The paper will include the improvement of portable sensing platforms
sensors displayed improved electrochemical properties after the based on graphene and graphene related materials, as well as
deposition of rGO onto the electrode since it increases the active simultaneous multiplexed detection of analytes, as already
sites of the electrode surface, promoting higher electron transfer occurred for CNT biosensors [1]. The fabrication of graphene sen-
[105]. Although the sensor displayed a wide linear response sors and biosensors as point-of care devices, which could lead to
(5e200 ppb) for detection of Pb2þ with high sensitivity, minimal better in situ clinical diagnosis or as in situ sensing platforms for
interferences of Cd2þ (<50 ppb) and Cu2þ (<100 ppb) were verified. environmental analysis should also be performed in future
Nitrite was also detected by a sensitive electrochemical sensor research. In addition, the combination of graphene with MIP should
based on rGO and Fe3O4 magnetic nanoparticles [106]. A large value be more studied in order to improve the analytical performance of
of heterogeneous electron transfer constant was obtained, which applied sensors and biosensors, taking advantage of the direct
indicated the high ability of rGO as electron carrier for the pro- contact of grapheneeMIP and the analyte. The fabrication of other
motion of electron transfer between the Fe3O4 nanoparticles and composites based on graphene and elements such as nanoparticles
the electrode surface. The sensor was applied to real samples of and organic polymers have been identified as an improvement of
sausage in order to demonstrate the good performance of the recent graphene based sensors and biosensors, and the research
sensor and it was found that there is a good agreement between the should be directed accordingly in order to improve their analytical
values obtained by the sensor and the reference method, and also performance, mainly in terms of reproducibility and limit of
between the spiked and found values [106]. Carbofuran and detection, as well as real-time analysis.
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 Acknowledgements
enhancement of the charge transfer efficiency. Fruits and vegeta-
bles (grapes, oranges, tomato, and cabbages) were used as real This work was funded by Portuguese Science Foundation (FCT)
samples in order to evaluate the practical application of the fabri- through scholarships (ref. SFRH/BPD/95961/2013) under POCH
cated electrochemical sensor. The satisfactory recoveries funds, co-financed by the European Social Fund and Portuguese
(96e104%) confirmed the stable and sensitive sensor for real National Funds from MEC. This work was also funded by national
sample analysis, and a good accuracy was also reported since the funds through FCT/MEC (PIDDAC) under project IF/00407/2013/
analytical results of the sensor were in agreement with those ob- CP1162/CT0023. Thanks are also due, for the financial support to
tained with a reference method (HPLC) [107]. CESAM (UID/AMB/50017), to FCT/MEC through national funds, and
the co-funding by the FEDER, within the PT2020 Partnership
3. Conclusions and future perspectives Agreement and Compete 2020.

This review reports recent works on the application of graphene


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