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Biomedical Applications of Graphene Nanomaterials and Beyond
Krishanu Ghosal and Kishor Sarkar*
Gene Therapy and Tissue Engineering Lab, Department of Polymer Science & Technology, University of Calcutta, 92 A.P.C. Road,
Kolkata 700 009, India
ABSTRACT: Graphene nanomaterials have been considered as a novel class of
nanomaterials that show exceptional structural, optical, thermal, electrical, and mechanical
properties. As a consequence, it has been extensively studied in various elds including
electronics, energy, catalysis, sensing, and biomedical elds. In the previous couple of
years, a signicant number of studies have been done on graphene-based nanomaterials,
where it is utilized in a wide range of bioapplications that includes delivery of small
molecule drugs/genes, biosensing, tissue engineering, bioimaging, and photothermal and
photodynamic therapies because of its excellent aqueous processability, surface
functionalizability, outstanding electrical and mechanical properties, tunable uorescence
properties, and surface-enhanced Raman scattering (SERS).Therefore, it is necessary to
get detailed knowledge about it. In this review, we will highlight the various synthesis
procedures of graphene family nanomaterials including graphene oxide (GO), reduced
graphene oxide (rGO), and graphene quantum dots (GQDs) as well as their biomedical
applications. We will also highlight the biocompatibity of graphene nanomaterials as well as its possible risk factors for
bioapplications. In conclusion, we will outline the future perspective and current challenges of graphene nanomaterials for
clinical applications.
KEYWORDS: grpahene, nanomaterials, biomedical application, biosensor, drug delivery
In the last few decades, nanoscience and nanotechnology have
been developed as a new prospect across many scientic
disciplines because of their specicoptoelectronicand
physicochemical properties. Invention of nanoscience and
nanotechnology is one of the largest revolutions since the
beginning of modern science and technology.
In this context,
carbonaceous nanomaterials like graphene, carbon dot,
graphene quantum dots (GQDs), graphene oxide (GO), and
reduced graphene oxide (rGO) have attracted much attention
over the last decades owing to their unique properties such as
large surface area, superior mechanical properties, and
exceptional thermal or electrical conductivity and optical
Graphene is a one-atom-thick 2D carbonaceous nanoma-
terial. It contains sp2-hybridized carbon atoms with honey-
comb structure. Since the revolutionary discovery by Geim and
Novoselov in 2004, graphene has emerged as a new eld of
research because of its unique properties.
These one-atom-
thick single-layer graphene sheets have several unique
properties, like very high electron transport capability at
room temperature,
high elasticity
and thermal conductivity,
high mechanical strength,
tunable optical properties,
quantum Hall eect at room temperature,
and tunable
band gap.
Unlike graphene, several fascinating properties also
showed by double-, few-, and multilayer sheets of graphene.
Subsequently, graphene is a conductive transparent nanoma-
terial, with low cost and substantial green environmental
impact, which makes it suitable for catalysis,
drug delivery,
and electrical and bioelectronics
Recent progress in the biomedical applications
of graphene nanomaterials already provides us some of the
sustainable biomedical devices such as deep brain stimulators
and blood glucose sensors.
Besides these biomedical
devices, graphene nanomaterials also have been used in tissue
gene therapy,
cell imaging,
and bioelec-
Although many successful implementations of
graphene nanomaterials have already been done in the eld
of physics, chemistry, and biology, it is an extremely hot topic
of research for researchers with many applications yet to come
in near future.
In this concern, a versatile range of nanomaterials including
nanoparticles, quantum dots, nanowires, and nanosheets have
emerged in the past few years, with extensive progress in
synthesis, characterization, and processing. One of the main
reasons to evaluate these eorts is how the size, composition,
and structure of these nanomaterials lead to novel optical,
electronic, mechanical, thermal, and magnetic properties.
These enhanced and extraordinary physical and chemical
properties of such nanomaterials open up exclusive oppor-
tunities in the eld of biology.
In this review, we mainly discuss biomedical applications of
graphene family nanomaterials, as well as their synthesis
procedure, biocompatibility, and biomedical applications
including biosensors, bioimaging, tissue engineering, drug
Received: March 28, 2018
Accepted: June 29, 2018
Published: June 29, 2018
Cite This: ACS Biomater. Sci. Eng. 2018, 4, 26532703
© 2018 American Chemical Society 2653 DOI: 10.1021/acsbiomaterials.8b00376
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delivery, gene therapy, photodynamic and photothermal
therapy, and their future perspective.
Graphene mainly has four forms, which are graphene itself, the
oxidized form of graphene, i.e., GO, the reduced form of
graphene oxide, i.e., rGO, and graphene quantum dots, i.e.,
GQD, which has a size less than 20 nm. GO is the oxidized
form of chemically modied graphene and it can be
synthesized by rapid oxidation of crystalline graphite followed
by some dispersion methods or sonication of colloidal
suspensions of graphite oxide in a wide variety of organic
whereas high temperature under reducing con-
ditions will convert GO to reduced graphene oxide. In this
section, we mainly focus on the relevant synthesis procedures
of these nanomaterials.
2.1. Synthesis of Graphene. There are several reported
synthesis procedures for the synthesis of graphene, including
mechanical exfoliation,
chemical vapor deposition (CVD),
plasma-enhanced chemical vapor deposition (PE-CVD),
cleavage of natural graphite,
hydrogen arc discharge,
epitaxial growth of electrically insulating material like boron
solution-processable methods,
and the micro-
wave synthesis method (see Figure 1).
Graphene was rst synthesized by mechanical exfoliation in
This simple cost-eective synthesis procedure created
an explosive growth in the research eld of graphene and
consequently demands graphene akes, which are valuable for
research to clarify graphene properties. But unfortunately,
graphene akes are usually available in few micrometers in this
method with irregular shapes due to their deterministically
uncontrolled azimuthal alignment. To overcome this problem,
researchers developed the CVD technique to synthesize
graphene akes in large-scale production. CVD is generally
carried out from carbon-containing gases in a catalytic
converter, where metal surfaces act as a catalyst, or by surface
segregation of carbon at high temperature and then dissolved
in bulk of metals like Ni, Cu, etc.
The rst interpretation of single-layer graphite was reported
by May in 1969.
Since then, a lot of research experiments
have been performed to synthesize single- or few-layer graphite
by surface segregation of carbon throughout the annealing
period of various carbon doped metals, e.g., Fe, Pt, Co, Ni, Pd.
A study done by Yu and his co-workers showed that an
atomically cleaned single-crystal Ni (111) surface in absence of
grain boundaries can produce more uniform and thinner few-
layered graphene, whereas polycrystalline Ni with grain
boundaries formed multilayer graphene. Here, grain bounda-
ries act as a nucleation site. They also noticed that dierent
cooling rates substantially aects the quality, number of
defects, and thickness of graphene. Another very important
observation they reported is that annealing of the Ni surface in
2atmosphere before graphene synthesis produces more
uniform graphene lms. They explained the reason behind this
phenomenon: that hydrogen removes impurities like S and P
that cause local alteration of carbon solubility in metals and
aecting graphene thickness.
Land and his co-workers used
hydrocarbon decomposition on Pt (111) surface to synthesize
single-layer graphite,
whereas ambient pressure CVD on
polycrystalline nickel produced 112 layer graphene lms.
By using CVD technique, Colombo and his co-worker
synthesized large-area graphene lms on the order of
centimeters on the copper surface using methane as s source
of carbon.
The reason behind this phenomenon is explained
as being based on dierential solubility of carbon in metal. The
solubility of carbon in nickel much greater than copper
(solubility of C in Ni is 1.3 at % at 1000 °C,
whereas in the
case of copper, it is less than 0.001 at % at 1000 °C
Similarly, large-area monolayer and multilayer graphene lms
were synthesized by Chen et al., specically bilayer graphene
lms on commercial CuNi alloy foils, by CVD using
hydrogen and methane as precursors. They also noticed that
the quality and thickness of graphene obtained on CuNi foils
substantially dependent on decomposition temperature and
cooling rate.
Graphene can be also prepared by surface
segregation of C atoms under ultrahigh vacuum at 1400 K on a
Ru(0001) surface
and by low pressure CVD on an Ir(111)
In another work, Ruoand his co-workers
discovered that the single crystals graphene can be synthesized
by low-pressure CVD technique on a copper foil.
Figure 1. (1) SEM images of hydrogen arc discharge-exfoliated graphene sheets (GS): (a) GS with a transparent wormlike morphology, (b) top
view, and (c) side view of the GS, (d) magnication of a part of the GS in panel c. Reproduced with permission from ref 5. Copyright 2009
American Chemical Society. (2) Morphological changes of 6HSiC(0001) during graphene growth: (a) Initial surface after H-etching imaged by
AFM. The step height is 15 Å. (b) AFM image of graphene on 6H-SiC(0001) with a nominal thickness of 1 ML formed by annealing in UHV at a
temperature of about 180 °C. (c) AFM image of graphene on 6HSiC(0001) with a nominal thickness of 1.2 ML formed by annealing in Ar (p=
900 mbar, T= 1650 °C). (d) LEEM image of a sample equivalent to that of c revealing macro-terraces covered with graphene up to 50 μm long
and at least 1 μm wide. Reproduced with permission from ref 36. Copyright 2009 Nature Publishing Group.
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Table 1. Comparison between Various Graphene Synthesis Methods
synthesis methods starting materials advantages disadvantages applications refs
mechanical exfoliation highly oriented
pyrolytic graphite very simple process, large scale production, good structural and electronic
quality low yields, time-consuming fundamental research
epitaxial growth on silicon
carbide 6H-6SiC wafer scale production, high qualities low yields, very high temperature
requirement, high synthesis cost electronics applications
reduction of graphite oxide graphite low cost, high yields, very good processability structural defects optoelectronics, composite
chemical vapor deposition on
transition metals hydrocarbon gas uniform, high quality, large scale production high cost, complicated technique, high
temperature requirement fundamental research and
electronic applications
reduction of ethanol by Na ethanol and sodium low cost, gram scale production, starting material other than graphite impure low quality graphene, violent
reaction composite materials applications
conversion of nanodiamond nanodiamond simple process high manufacture cost, high temperature
requirement composite materials applications
organic synthesis polyacyclic
hydrocarbons good structural quality, very good processability dicult synthesis process, limited size range electronics and optoelectronic
arc discharge of graphite graphite low cost, one step synthesis procedure impure graphene, nonuniform composite materials applications
electron beam irradiation on
PMMA nanobers PMMA low-temperature process, controlled way to produce graphene nonuniform, yield is low electronics and optoelectronic
unzipping of carbon nanotubes carbon nanotubes low-cost, very simple process, large-scale production, graphene nanoribbons
with controlled edges and widths time consuming process electronics applications
thermal reaction of polyaromatic
hydrocarbon polyaromatic
hydrocarbons wafer scale, low-cost production high-temperature requirement, nonuniform electronics and optoelectronics
liquid-phase exfoliation graphite low-cost, large-scale production, direct synthesis procedure impurity present in nal product, time-
consuming process transparent electrodes, electronic
transfer batch fabrication acetic acid, CH4,H
2uniform single-layer graphene, good carrier mobility, electrical and
mechanical continuation over a large distance complicated, high temperature required electronics applications
molecular beam deposition acetylene large-area, high-quality graphene, good control over process high temperature required electronics applications
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Although exfoliated graphene akes exhibit electrical trans-
port properties like CVD based graphene but when it comes to
single graphene domain, grain boundary plays an important
role. This is the main reason behind the variability of electron
mobility that is frequently observed in CVD-based graphene.
In addition to the CVD technique, another technique, i.e.. the
epitaxial growth technique, is also used to produce large-scale
monolayer graphene where graphene is grown on a single-
crystal silicon carbide (SiC) wafer by vacuum graphitization.
Compared to the above techniques, the epitaxial growth
method simply provides the fabrication of low-defect-density,
large-scale graphene lms on a semi-insulating SiC surface and
it can be used without transfer to any insulating substrate.
the year of 1978, Tairov and Tzvetkov developed a modied
sublimation growth process for 6H-SiC.
A few years earlier in
1975, Tooren and his group showed that segregation of silicon
from SiC (0001) single crystal leads to the formation of a
graphite layer on the SiC surface. They also noticed that under
ultrahigh vacuum (<1 ×1010 Torr) and at elevated
temperature, Si-face consist with monocrystalline graphite
layer, whereas C-face consists with polycrystalline graphite
Graphene formation starts on the top of the SiC surface and
proceeds inwards. One graphene layer is formed from
decomposition of approximately three SiC bilayers. Initially,
a carbon-rich surface layer also called buer layer reconstruc-
tion starts where carbon atoms situated isostructurally to the
graphene; however, there are neither sp2structure nor covalent
bonds attached to underlying Si atoms. This silicon layer acts
as an insulator and does not show graphene-like electronic
properties. It is also observed that the sublimation rate of SiC
is reduced drastically, when an inert gas atmosphere (up to 1
bar) is introduced, and graphene starts to grow at temperature
more than 14001500 °C, at this temperature carbon atom
form high-quality graphene (only 12 layers) due to greater
mobility of C atoms.
Synthesis of graphene by reduction of
graphite oxide is another easy route of graphene synthesis. Wei
and his co-workers demonstrated a green and sustainable
approach for the synthesis of graphene.
They synthesized
graphene nanosheets from exfoliated graphite oxide by Fe
reduction. Fe eliminates, oxygen functionalities from graphite
oxide and produce graphene. This technique oers a large
scale, eco-friendly and cost-eective production of graphene. In
2008 Mullen and his group discovered another novel approach
for synthesis of 2D graphene nanoribbons.
They produced
graphene nanoribbons with the help of Suzuki-Miyaura
coupling reaction using 1,4-diiodo-2,3,5,6-tetraphenylbenzene
and 4-bromophenylboronic acid as a starting material. They
developed graphene nanoribbons up to 12 nm in length, and
further microscopic studies also conrmed that these nanorib-
bons have a high tendency to self-assemble.
nanoribbons were also synthesized by electron beam
irradiation on poly(methyl- methacrylate) nanobers. This
low-temperature, one-step synthesis process provides a
designed and controlled way to obtain graphene.
and their group manufactured graphene from graphite by
liquid-phase exfoliation technique. In this process, they rst
dispersed graphite in organic solvents followed by exfoliation.
Recently, another large-scale graphene synthesis process was
developed by Stride research group.
They synthesized
graphene from ethanol reduction by sodium followed by
Recently, solution-processable synthesis of graphene has
gained tremendous attraction because of its several advantages
like eco-friendliness, scalability, and easy synthesis method
compared to CVD or epitaxial growth or hydrogen arc
discharge. In this concern, Wallace and co-workers reported for
the rst time solution-processable synthesis of graphene sheets
in large scale. They synthesized graphite oxide from graphite
by a modied Hummers method. They then puried the
graphite oxide by dialysis followed by dispersion using
ultrasonication to get GO. After that, they removed the
unexfoliated graphite oxide by centrifugation and nally the
GO dispersion treated with hydrazine and ammonia solution
to get processable graphene sheets. During the reaction they
maintained the hydrazine to GO ratio at about 7:10, which is
optimal for synthesis.
Shams et al. synthesized solution
processable few layer graphene nanosheets from dead camphor
leaves. They used a greener approach for the synthesis of
graphene sheets. For the synthesis they used dirt free vacuum-
dried camphor leaves as a starting material and heated in a
vacuum oven under nitrogen atmosphere up to 1200 °Cat10
°C/min and maintained for 4 min. After that they cooled the
product and mixed with D-tyrosine and trichloromethane with
sonication for 15 min followed by centrifugation to precipitate
large carbon particles to acquire the few layer graphene
In a recent work, Verramani et al. also reported the
synthesis of a solution-processable graphene sheetlike porous
activated carbon (GPAC). For the preparation of GPAC, they
cut the Bougainvillea glabra owers into small pieces, washed
with water, and dried in an oven at 100 °C. They then
preheated the dried material at 200 °C for 6 h. After that, they
carried out activation process where they mixed the preheated
carbon powder with 10% ZnCl2and stirred at 60 °C under a
N2atmosphere for 24 h followed by carbonization in a tubular
furnace for 3 h in a N2atmosphere. Finally, they washed the
carbonized product with HCl and distilled water for
neutralization and drying.
The microwave synthesis method is another newer synthesis
process for graphene. Hazmi and co-workers reported the
synthesis of monolayer, bilayer, and multilayered graphene
using precise microwave power and temperature. In detail, they
treated graphite akes with ice cold glutaric acid to separate
out graphite akes at a distance equal to the glutaric acid
molecule (0.88 nm). After that, they dispersed the pretreated
graphite akes in methanol and introduced in Teon autoclave
and nally placed into a microwave oven. The microwave
power, time, and reaction temperature determine the faith of
the graphene layers.
In addition to these, there are some other graphene synthesis
methods having various advantages and disadvantages that are
listed in Table 1.
2.2. Synthesis of Graphene Oxide (GO) and Reduced
Graphene Oxide (rGO). One of the most signicant
branches of graphene research concerns with GO and rGO.
GO contains many oxygen functionalities, as it is prepared by
oxidation of graphite and reduction of graphene oxide produce
reduced graphene oxide. Until now there have been three well-
known methods for synthesis of GO, Boride,
and Hummers methods,
or by slight modications of
these procedures. In all these procedures, oxidation of graphite
involves at dierent level. Boride performed a reaction
involving a mixture of nitric acid (HNO3) and potassium
chlorate (KClO3) to oxidize graphite and produced graphite
Nearly four decades later, Staudenmaier modied and
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improved Borides method by adding several aliquots of
chlorate during the course of the reaction. Staudenmaier also
added some concentrated sulfuric acid to increase the acidity
of the reaction medium. This slight modication in the
procedure resulted in overall oxidation of graphite similar to
Borides method but performed more eciently in a single
reaction vessel.
Sixty years later, Hummers developed
another alternative method to synthesize GO by reacting
graphite with sodium nitrate (NaNO3), concentrated sulfuric
acid (Con H2SO4), and potassium permanganate (KMnO4).
Apart from these well-known methods, researchers have
developed a few modied methods such as the addition of
one or two additional oxidizing agents during synthesis steps to
synthesize GO based on previous methods as discussed earlier;
however these three methods remain the main methods for
synthesis of GO.
Although it has been observed that the nal
products of these reactions show a wide range of variance
depending not only the specic oxidants used but also depends
on reaction conditions of the medium and graphite source.
Both Boride and Staudenmaier treated with potassium chlorate
(KClO3) and concentrated nitric acid (con HNO3) for their
method. Nitric acid is one of the most common oxidizing
agents used for chemical reactions; it also reacts signicantly
with aromatic carbon surfaces and forms various oxide
containing species like ketones, lactones, carboxyls, etc.
In the case of Hummers method, a mixture of potassium
permanganate and sulfuric acid is used. Potassium permanga-
nate reacts with sulfuric acid and form dimanganeseheptaoxide
(Mn2O7), this bimetallic heptaoxide is much more reactive
than its monometallic tetraoxide analogue and it is well-known
that this bimetallic heptaoxide detonates when placed in
contact with organic compounds or when heated temperature
higher than 55 °C.
Tour and his group developed an
improved GO production method based on Hummers
They found that excluding NaNO3, if the amount
of KMnO4is increased and a mixture of 9:1 H2SO4/H3PO4are
used then it improves the eectiveness of the oxidation process
as shown in Figure 2. Polar oxygen group, that present in GO
provide it good hydrophilicity, so it can be exfoliated in many
polar solvents, particularly in water. GO oers a wide range of
possibilities for the manufacture of the chemically modied
graphene in industrial scale.
Other than the Hummers and modied Hummers methods,
researchers recently came with some alternative rapid and
improved synthesis methods, like a microwave synthesis
method for the preparation of GO. As an example, Wang et
al. synthesized GO nanosheets by microwave irradiation. Their
synthesis method involved conc. H2SO4, graphite akes, and
KMnO4. They mixed it in a three -necked ask and placed it in
a microwave oven for 150 s in a cycle of 30 s. After that, they
stopped the reaction by adding 15 mL of 30% H2O2followed
by centrifugation and discarded the supernatant to get GO.
Finally, they ultrasonicated the mixture to get a homogeneous
suspension of GO.
He and co-workers also reported one pot,
one step microwave assisted synthesis of GO with a yield of
120%. Unlike previous work, they used conc. HNO3along with
conc. H2SO4, graphite akes, and KMnO4for the preparation
of GO. Under microwave irradiation of 300 W for 40 s,
graphite akes turned into GO.
In addition to the microwave
synthesis method, Grossman and co-workers reported another
improved synthesis method for preparation of GO.
they demonstrated that a mild thermal annealing procedure
(5080 °C) can enhance the sheet properties in GO produced
by Hummers method in a large scale without aecting the
oxygen content preserved and without involving any other
chemical treatment. The thermal annealing facilitate to
transform the mixed up sp2sp3hybrid phases into a distinct
oxidized and well-ordered graphitic phases which resulted in
improved optical and electronic properties than as synthesized
GO produced by Hummers method. Yamaguchi et al. also
revealed that oxygen functionalization of GO can be controlled
by thermal annealing, which produces GO with enhanced
electronic properties.
To date, only a few preparation methods have been reported
for reduced graphene oxide (rGO). Most researchers used
hydrazine hydrate for synthesis of rGO from GO.
However, some other reducing agents also used for the
preparation of rGO. Paredes and his group compared eect of
dierent reducing agents on graphene oxide. They used
ammonia, potassium hydroxide, sodium borohydride, pyro-
gallol, ascorbic acid(vitamin-C) and hydrazine monohydrate as
reducing agents and reached in a conclusion that ascorbic acid
is the ideal substitute for hydrazine for reduction of the GO.
Figure 2. Representation of the synthesis procedures of GO followed starting with graphite akes (GF). Under-oxidized hydrophobic carbon
material recovered during the purication of IGO, HGO, and HGO+. The increased eciency of the IGO method is indicated by the very small
amount of under-oxidized material produced. Reproduced with permission from ref 73. Copyright 2010 American Chemical Society.
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Lee and his co-workers are used NaBH4for reduction of GO
and found that, the electrical resistance of rGO reduced by
NaBH4is much lower than reduced by hydrazine. They
proposed that it might be formation of CN groups in case of
hydrazine, which can act as donors, recompensing the hole
carriers in rGO but on the other hand in case of NaBH4
reduction the interlayer spacing between two graphite oxide
layer expanded to some extent by the formation of transitional
boron oxide complexes and then contracted by subsequent
removal of hydroxyl and carbonyl groups with boron oxide
Cheng research group reported a unique method for
reduction of GO.
They used hydrohalic acid for reduction
of GO. They demonstrated that hydrohalic acid can reduce
GO very eectively at 100 °C for 1 h without destroying their
exibility and integrity images are shown in Figure 3. They also
checked electrical conductance of the produced rGO and
gure out that its electrical conductance is much better than
other rGO produced by other methods. Other than these
studies, some research groups tried to understand the
theoretical framework of the oxygen bonds during the
reduction process of GO to rGO. Such as, Kumar et al.
investigated the impact of oxygen clustering on the thermal
reduction of graphene oxide.
They found that the number of
oxygen and carbon atoms can be altered during the synthesis of
rGO, depending on the degree of oxygen clustering without
hampering the reduction temperature. Shen et al.
and Niu et
also tried to reveal the eect of low-temperature thermal
treatments during the synthesis of rGO from GO. Shen et al.
demonstrated that during the low-temperature thermal
reduction process that the decomposition of functional groups
present in GO occurs in 4 distinct steps ranging from below
160 °C to above 300 °C,
whereas Niu et al. demonstrated
that rGO can be prepared eectively by thermal treatment of
GO at 200 °C.
To date, these methods have been the most
signicant and well-known methods for the synthesis of
graphene oxide (GO) and reduced graphene oxide (rGO).
2.3. Synthesis of Graphene Quantum Dots (GQDs).
The latest member of the graphene family is graphene
quantum dots (GQDs) ,which are smaller in size, ranging
from 3 to 20 nm
with sp2sp2carbon bonds similar to
graphene and possessing large surface area, and better surface
grafting using ππconjugation and surface functionalization.
In addition to the above properties, GQDs exhibit new
phenomena due to quantum connement and edge eects that
are similar to C-dots. Typically, GQDs contain hydrophilic
functional groups such as hydroxyl and carboxylic acid at the
edge, which are similar to graphene or graphene oxide, thus
imprinting them with excellent water solubility and sub-
sequently allowing them to be modied with various organic,
inorganic, and biological species according to requirement.
Because of these superior properties, GQDs have been
considered as one of the most promising nanomaterials and
signicant research eorts are being put toward synthesizing
low-toxicity GQDs using eco-friendly alternative methods,
which is a major concern with other quantum dots (CdSe, PbS,
CdS, etc.). In this regard, there are mainly two approaches to
synthesize GQDs, namely, the top-down method and bottom-
up method. Top-down method includes chemical ablation
from graphene and electrochemical exfoliation. Bottom-up
approaches consists of cage opening of fullerene and solution
chemistry synthesis, microwave synthesis etc. Below, we shall
do a small discussion about various synthesis methods of
2.3.1. Top-down Methods. Chemical ablation from
graphene is one of the most common GQD synthesis
techniques. It includes hydrothermal, solvothermal, hydrazine
hydrate reduction method, etc.
In 2010, Wu and his co-workers synthesized GQDs from
graphene oxide sheets by hydrothermal method where large
Figure 3. (1) Optical photographs of the reducing process by immersing a GO lm into dierent reducing agents for dierent times at room
temperature. (a) Three liquid reducing agents: 50 mM NaBH4aqueous solution (NaBH4), 85% N2H4·H2O solution (N2H4), and 55% HI acid
solution (HI). (bd) GO lms reduced by the three agents for 10 s, 10 min, and 16 h. (eg) Enlarged views from b, which show that the
phenomenon occurred after 10 s immersion of GO lms to the three liquid reducing agents. (2) Optical photographs and mechanical properties of
the GO lms reduced by dierent chemical agents. (a) As assembled GO lm. (b) 1 h HI acid-reduced GO lm at 100 C. (c) Stressstrain curve
of the GO lm and HI acid-reduced GO lm. (df) Hydrazine vapor-, N2H4·H2O, and NaBH4-reduced GO lms. (df) Scale bar is 5 mm.
Reproduced with permission from ref 84. Copyright 2010 Elsevier.
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graphene oxide sheets were cut into small one by controlled
oxidation in a mixture of sulfuric acid and nitric acid under
mild ultrasonication. The oxidized small graphene sheets were
then reduced to GQDs under hydrothermal condition at a
temperature of 200 °C for 10h.
Shen et al. reported GQDs
surface passivation with polyethylene glycol by hydrothermal
reaction, using small GO sheets and polyethylene glycol as a
starting material.
Yang group made green uorescent GQDs
by solvothermal method with PL quantum yield as high as
11.4% and average diameter of 5.3 nm and height of 1.2 nm,
from which they concluded that most of the GQDs are single
or bilayerd.
In addition to this, GQDs were also synthesized
by hydrazine hydrate reduction method from small graphene
oxide sheets. Zhu and his group synthesized GQDs by
hydrazine hydrate reduction of GO with their surface
passivation by PEG diamine.
For this method, at rst they
oxidized GO with HNO3cut into small pieces, then these
small GO sheets were passivated with PEG diamine and nally
reduced by hydrazine hydrate to get the GQDs. The diameter
of the synthesized GQDs lies between 5 and 19 nm, suggesting
that the formation of monodisperse GQDs. Prior to this work,
Chhowalla and co-workers discovered that uorescence
intensity of the GO thin lm was changed after each
incremental when it is exposed to hydrazine-vapor (from 20
s up to 60 min).
Pangs group synthesized GQDs by electrooxidation of
graphite in aqueous solution (see Figure 4). They electro-
chemically oxidized a graphitic at 3 V against a saturated
calomel electrode with a Pt wire counter electrode in a
NaH2PO4aqueous solution.
Chi group also prepared GQDs
They performed the experiment in an
electrochemical cell consisting a graphite rod as working
electrode, a Pt mesh as a counter electrode and Ag/AgCl
electrode as reference electrode at pH 7.0 phosphate buer
solution. Li et al. reported an electrochemical synthesis
procedure for green-luminescent functional GQDs with a
uniform size of 35 nm. GQDs were formed by electro-
chemical oxidation of graphene lm electrode in phosphate
buer solution. Due to oxygen-containing functional groups on
its surface it gained high stability in the aqueous medium. The
aqueous dispersion of GQDs are stable up to 3 months.
Qu and co-workers reported a simple electrochemical
approach for N-doped GQDs with oxygen group functional
They used graphene lm as a working electrode, Pt
wire and Ag/AgCl act as a counter and reference electrode.
The electrolyte was 0.1 M TBAP in acetonitrile. The
synthesized N-GQDs have uniform diameter of 25nm
with a topographic height of 12.5 nm suggesting that each N-
GQD consist of 15 graphene layers.
2.3.2. Bottom-up Methods. The Loh group reported a
mechanistic approach for synthesis of a series of atomically
dened GQDs by metal catalyzed of fullerene. They
synthesized dierent geometrically shaped GQDs such as
triangular, parallelogram, hexagonal, and trapezoid-shaped on a
Ru surface using C60 molecules as a starting compound.
Although the formation of dierent-shaped GQDs was
temperature dependent. They suggested that GQDs are
formed through the opening of C60 cage on ruthenium surface
through the strong interaction between Ru surface-carbon
atoms of C60.
The fragmentation of embedded fullerene molecules at
elevated temperature then produces carbon cluster and
subsequently under GO diusion and aggregation to form
Chua et al. also synthesized ultrasmall graphene
quantum dots by cage opening of Buckminster fullerene. For
synthesis fullerene and sodium nitrate were stirred with sulfuric
acid. Then the mixture was cooled to 0 °C temperature after
that potassium permanganate (7.5 g) was added for a period of
2 h. The mixture was then cooled and treated with H2O2to
remove the unreacted potassium permanganate and manganese
dioxide followed by neutralization with NaOH. The size of the
GQDs are 35 nm as depicted in Figure 5.
Among other
bottom up methods it is the most common approach for
synthesis of GQDs. Li and co-workers synthesized large scale
graphene quantum dots by a solvothermal method using
dendritic arene precursors. Their as-prepared GQDs were
made with graphene moieties containing 168, 132, and 170
conjugated carbon atoms.
Mullen and his co-workers
reported multicolour photoluminescent GQDs with a uniform
size of 60 nm diameter and thickness of 23 nm using
unsubstituted hexa-peri-hexabenzocoronene as the precur-
Zhao and his co-workers synthesized GQDs with an
Figure 4. (a) Blue uorescence of N-GQDs upon UV light exposure.
(b) Schematic structure of the GQDs. (c) TEM images of N-GQDs.
Reproduced with permission from ref 98. Copyright 2012 American
Chemical Society.
Figure 5. Illustration of the oxidation and cage-opening of fullerene
C60 with treatment of strong acid and chemical oxidant. On the right
side below luminescence of graphene QDs excited with a blue laser
pointer (405 nm). Reproduced with permission from ref 99.
Copyright 2015 American Chemical Society.
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average diameter of 4.66 ±1.24 nm by solution chemistry
pyrolysis using L-glutamic acid as a precursor.
The quantum
yield of the synthesized GQDs as high as 54.5%.
Recently, Hu and co-workers prepared strong blue
uorescence emitting GQDs having 14% quantum yield
using aspartic acid and NH4HCO3as precursor with the
help of microwave mediated pyrolysis technique.
On the
other hand, Zhu and his team also reported the synthesis of
GQDs having 11.7% quantum yield and greenish-yellow
luminescence from GO nanosheets but they obtained GQDs
by simultaneous cleaving and reduction of GO nanosheets
under acidic condition without using any external reducing
agent. The diameter of the synthesized GQDs lies between 2
and 7 nm with an average diameter of 4.5 nm. The topographic
height of the GQDs were mostly between 0.5 and 2 nm with
an average height of 1.2 nm, suggesting that most of the GQDs
are single or bilayered.
Since the revolutionary discovery of graphene, graphene
nanomaterials have gained tremendous attention in all the
elds of science and technology. Until now, dierent types of
graphene nanomaterials have been devolved in terms of their
sizes, shapes and surface functionalization which endow them
with dierent physical chemical and biomedical characteristics.
So in vitro cytocompatibility investigations are very essential in
order to develop graphene-related biomedical devices/
materials as well, as it is very crucial for further in vivo studies
of graphene nanomaterials.
Cytocompatibility of graphene and graphene based nano-
materials with living cells and microorganisms play a crucial
role for bioapplications. More specically the cytocompatibility
of graphene nanomaterials signicantly depends on their
physical and chemical properties (such as hydrophobicity,
number of graphene sheets, size) and concentration.
hydrophilic and nanosize graphene forms (e.g-GO, GQDs) are
found to be less toxic compared to that of hydrophobic and
large size graphene. As an example, Cui and co-workers
reported that in the presence of GO, human broblast cells
decreases the viability slightly (less than 20% when exposed for
4 days) at a concentration of less than 10 μg/mL; however,
higher concentration and longer exposure time lead to a
signicant amount of cell death. They found that GO
concentrations more than 50 μg/mL are obviously cytotoxic
even after only 1 day of exposure (>20%).
The possible
explanation of this phenomenon is due to the formation of
agglomeration in physiological medium through ππinter-
actions between the GO layers. As a result, aggregated
macroparticles are failed to enter into the cells and entrapped
cytoskeleton, membrane deformation and increase in inter-
cellular stress which consequently leads to cell death. These
factors also applicable for other graphene family nanomaterials.
So in order to get good and long-term stability as well as low
Figure 6. Enhanced neural-dierentiation of hNSCs on graphene lms. All scale bars represent 200 μm. (a) Bright-eld images of the hNSCs
dierentiated for 3 days (left), 2 weeks (middle), and 3 weeks (right). Note that the hNSCs on glass were gradually retracted and detached after 2
weeks, whereas those on graphene remained stable even after 3 weeks of dierentiation. (b) Bright-eld (top row) and uorescence (bottom row)
images of hNSCs dierentiated on glass (left) and graphene (right) after one month dierentiation. The dierentiated hNSCs were immunostained
with GFAP (red) for astroglial cells, TUJ1 (green) for neural cells, and DAPI (blue) for nuclei. Note that more hNSCs were adhered to graphene
than to glass. (c) Cell counting per area (0.64 mm2) on graphene and glass regions after one-month dierentiation. Note that much more cells were
observed on graphene in comparison to the glass regions (n=5,p< 0.001). (d) Percentage of immunoreactive cells for GFAP (red) and TUJ1
(green) on glass and graphene. Note that glass regions show more GFAP-positive cells (glia) than TUJ1-positive ones (neurons), while graphene
regions have more TUJ1-positive ones (neurons) than GFAP-positive ones (glia) (n=5,p< 0.05). Reproduced with permission from ref 114.
Copyright 2011 Wiley.
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cytotoxicity for bioapplication purposes, the graphene nano-
materials should be stable in biological media without
formation of any aggregation, and it is already well-established
that proper functionalization of graphene nanomaterials can
increase its stability in biological media and consequently
increase the cytocompatibility. In this concern, Koyakutty and
co-workers reported that cytocompatibility of pristine
graphene can be increased signicantly when it is subjected
to carboxyl functionalization.
They discovered that 24 h
exposure by the carboxyl-functionalized graphene at a
concentration of 300 μg/mL did not signicantly aect the
viability of Vero cells. In another work, they studied the
cytocompatibility and immune response of both pristine
graphene and functionalized graphene.
They observed that
the macrophage cells (RAW 264.7) uptake was much higher
amount for functionalized hydrophilic graphene compared
with hydrophobic pristine graphene, which was mainly
retained at the cell surface and induces ROS-mediated
apoptosis above the 50 μg/mL concentration, whereas
functionalized graphene showed better cytocompatibility with
no stress eect up to 75 μg/mL concentration. However,
despite the cytocompatibility improvement of graphene
through hydrophilic functionalization, the reactive oxygen
species (ROS) production in mammalian cells by all forms of
graphene nanomaterials is a major drawback in biological
application. Therefore, care should be taken seriously when
using graphene materials for biomedical applications.
The synthesis methods of graphene on the hydrophilicity of
graphene and consequently its cytotoxic eects were also
found. For example, graphene synthesized by CVD increased
apoptosis, the level of lactate dehydrogenase, and generation of
ROS in neural cells.
Graphene nanomaterials, especially hydrophilic forms of
graphene (e.g., GO) can strongly promote cell adhesion. More
specically, a number of studies have been done where
graphene and its derivatives act as a substrate for various types
of cells including stem cells.
Hong and co-workers showed
the formation of neuron cells instead of glial cells through
dierentiation of human neural stem cell by laminin coated
graphene without using any biological cues
as shown in
Figure 6, where enhanced neural dierentiation observed in
the presence of graphene lms.
Chen et al. also exhibited the dierentiation of induced
pluripotent stem (iPSC) cells in the presence of graphene and
graphene oxide. They found similar behavior of iPSC toward
glass substrate and graphene with respect to cell adhesion and
dierentiation, whereas GO showed better adhesion and
dierentiation compared to that of bare graphene.
hydrophobicity, physical dimensions of the graphene-based
nanomaterials, especially size and number of graphitic layers,
also play a crucial role in determining their cytocompatibility.
The correlation of cytocompatibility with size was further
observed by Haynes and co-workers. They synthesized
dierent sizes of graphene and graphene oxide followed by
observation of their biological eects on human erythrocytes
and skin broblasts.
They found the hemolytic activity of
human erythrocyte cells by both graphene and GO in a dose-
dependent manner. Interestingly, it was found that smaller-
sized GO sheets showed very high hemolytic activity compared
to that of large-sized GO sheets. When compared between
graphene and graphene oxide sheets, GO sheets are dispersed
much better because of their functional groups, whereas
graphene tends to aggregate and showed much lower amounts
of hemolytic activity. In a recent study by Chen and co-
workers, where they demonstrated that the cell capture
capability of the functionalized graphene oxide nanosheets
can be enhanced by 5492% through oxygen clustering.
More specically, their system is highly sensitive toward Class
II MHC-positive cells from murine whole blood at room
temperature, which is almost double of the eciency oered
by other devices made directly using as-synthesized GO. In
addition to that, clustering of oxygen during the phase
transformation of GO improves substance adherence proper-
ties, which ultimately results in enhanced stem cell dier-
Size is another crucial factor that determines the
cytotoxicity of nanomaterials. In this context, Yue et al.
demonstrated the role of the lateral dimension (i.e., 350 nm
and 2 μm) of GO in terms of cellular responses and cell
They examined the ability to internalize GO in six
dierent cell lines and found that only two phagocytic cell lines
are capable of internalizing both types of GO. In comparison
with size-independent uptake, the intercellular phenomenon
and cytokine proles were signicantly aected by lateral
dimension, specically, microsized GO induced much stronger
inammation responses than nanosized GO; in other words,
nanosized GO demonstrated more cytocompatibility than
microsized GO. Fan and co-workers studied the uptake
mechanism and cytocompatibility of GQDs on human neural
stem cells (hNSCs).
From their result, they concluded that
the GQDs were internalized in to the hNSCs through
endocytosis pathway although the cellular uptake was
concentration and time dependent. Additionally, they found
no signicant change in the cell viability, dierentiation,
metabolic activity, and proliferation of hNSCs after treatment
with GQDs.
To access the full potential of graphene-based nanomaterials
for biomedical applications, it is necessary to know its
biocompatibility in vivo. At present, very few number of
studies have been highlighted on the in vivo biocompatibility
by graphene nanomaterials. Similar to cytocompatibility, in
vivo biocompatibity of graphene nanomaterials also depends
upon physicochemical properties (e.g., includes size, surface
functionalization), concentration biodegradation and another
very important parameter which does not present in case of in
vitro, that is route of administration.
Girish et al. addressed for the rst time the crucial issue of
biodegradability of pristine graphene by help of confocal
Raman imaging, which is very important criteria for any in vivo
biological applications of graphene.
Their study revealed
that time-bound spectral alterations such as increase in ID/IG
ratio, formation of defective Dband and widening of D and G
bands of graphene, embedded in dierent organs such as liver,
kidney over a time of 890 days. This is due to the increase in
structural disorders in graphene phagocytosed by macro-
phages. In the case of spleen bound samples most enhanced
amount of disorder was observed, which leads to complete
amorphization after 3 months of intravenous injection. Their
ndings suggest that the possible biodegradability of graphene
in vivo may have a greater impact on the practical applications
of graphene in the eld of biology and medicine. In vivo
toxicity study of graphene oxide (GO) showed that the
intravenous GO administration increased the accumulation of
GO largely in lung and liver for longer time although it was
ACS Biomaterials Science & Engineering Review
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The accumulation may be occurred due to
instability and nonspecic binding of GO with dierent
proteins. After injection, the blood ows to the lung initially
and hence results more accumulation of GO in lung compared
to other body organ. Singh et al. used GO and rGO to analyze
the eect on blood platelets.
They reported for the rst time
atomically thin GO sheets caused strong aggregatory responses
in platelets through activation of a family of Src kinases and
subsequent release of calcium from intercellular compartments.
Furthermore, they noticed that intravenous administration of
GO in mice induced extensive pulmonary thromboembolism.
This behavior was linked with the charge distribution on the
surface of GO as the aggregation properties were signicantly
decreased when it is converted to rGO.
Toxicity of graphene and graphene oxide can be reduced
signicantly in case of pristine graphene after liquid phase
exfoliation and further decreased when the unoxidized
graphene is well-dispersed with the block copolymer Pluronic
as shown in Figure 7. Mutlu and co-workers demonstrated that
covalent oxidation of graphene play a major role to its
pulmonary toxicity and they suggested that dispersion of
pristine graphene in Pluronic provides a pathway for the safe
handling and potential biomedical applications of graphene
Another in vivo biocompatibility study of
GO was done by Zhang et al., where they observed that
compared with other carbon nanomaterials, GO showed long
blood circulation time (half-time 5.3 ±1.2 h), and very little
uptake by reticuloendothelial system.
Also, no pathological
changes were observed in major organs, when mice were
exposed to 1 mg/kg body weight of GO for 14 days. In
addition to this, GO exhibited good biocompatibility with red
blood cells. The dierence in oxidation states of GO also plays
a crucial role to in vivo biocompatibility. In this context,
Langer and co-workers evaluated the biocompatibility of GO
with two dierent oxidation states followed by implantation in
subcutaneous and intraperitoneal tissue sites.
suggested that GO is moderately biocompatible in vivo in
both tissue sites, with the inammatory reaction in feedback to
implantation consistent with a typical foreign body reaction.
Notably, reduction in the degree of oxidation resulted in easier
immune cell inltration, uptake and clearance following the
both tissues implantation. Furthermore, the toxicity of
graphene nanomaterials can be reduced eectively through
functionalization of GO by nontoxic biomolecules or varying
the size of GO during the synthesis steps. Additionally, degree
of oxidation also plays an important role to determine the
toxicity of GO. In this concern, Liu and co-workers showed
that in vivo toxicity of GO can be modulated through chemical
For example, PEGylation of GO reduces toxic
eect in mice. In another study, they observed the eect of
long-term biodistribution of 125I labeled nanographene sheets
functionalized with polyethylene glycol and systematically
examined the toxicity over time. They found that PEGylated
NGS mainly accumulate in the reticuloendothelial system
including liver and spleen after intravenous injection and
gradually released by both renal and fecal excretion. PEGylated
NGS do not cause any signicant toxicity at a dose of 20 mg/
kg to the treated mice in a period of 3 months as veried by
hematological analysis, blood biochemistry, and histological
experiment. Researchers also explored the mechanism of stress-
induced toxicity of graphene oxide, modied with PEGylated
poly-L-lysine on C. elegans as an in vivo model.
On the basis
of their result, they proposed multiple mechanistic pathways
for the toxicity. When the nematode treated with a
concentration range of between 5 and 20 μg/mL no changes
were observed in means of longevity, impairment of
locomotion, cell wall damage and reproducibility but polymer
factionalized GO signicantly aect the resistance of
nematode. More specically under oxidative or heat stress
conditions and leading to death. The excessive amount of ROS
generation diminished the inherent antioxidant defense system,
thus provoking dramatic toxic eect on C. elegans under
pathophysiological condition. In conclusion, the toxicity of
graphene based nanomaterials in in vivo is mainly dependent
Figure 7. Aggregated graphene induces patchy brosis in mice. Mice
were treated with highly puried and dispersed preparations of
graphene in 2% Pluronic F 108NF (Dispersed), aggregates of
graphene in water (Aggregated), or GO in water (Oxide) by
intratracheal instillation and 21 days later, the lungs were examined
for markers of brosis. (a) Trichrome stained lung sections. (b) Sirius
Red-stained lung sections (bottom panels are photomicrographs
obtained using a polarizing lter). (c) Total lung collagen determined
by picrosirius red precipitation of whole lung homogenates (GD,
dispersed graphene; GA, aggregated graphene; GO, graphene oxide).
Representative images from four or more animals per group are
shown, N= 8 for picrosirius red precipitation, dierences between
groups are not signicant. Reproduced with permission from ref 123.
Copyright 2011 American Chemical Society.
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on the size, dose, exposure duration, as well as numbers of
graphene sheets. More signicantly, the surface properties of
graphene-based nanomaterialssuchasfunctionalization/
chemical structure play an important role in determining its
toxicity; size, shape, synthesis method, biodegradation, and
route of administration also play a very crucial role in
determining biocompatibility of graphene nanomaterials.
5.1. Biosensor. Because of the unique properties of
graphene family nanomaterials, they are extensively used as
biosensors. Recently, a number of electrochemical, uorescent,
and eld-eect transistor biosensors have been developed
based on the graphene nanomaterials by various research
groups as discussed below.
5.1.1. Electrochemical Biosensor. In the last few decades,
high specicity and sensitivity of graphene material have made
it a tremendously attractive compound as electrochemical
biosensors among other biosensors. The specicity of electro-
chemical biosensor lies on the fact that dierent molecules
oxidize or reduce at dierent potential window. In the case of
graphene-based electrochemical biosensor, electron transfer for
oxidation or reduction processes occurs between the graphene
and analyte molecules, and this heterogeneous electron transfer
process happens at the edges and corners of the graphene or at
the defect sites of basal plane.
The large surface area of
graphene provides enormous number of corners edges and
defects which can act as superior electroactive sites.
Graphene has been extensively used for glucose sensors
because diabetes has become a worldwide public health threat.
For glucose sensing, glucose oxidase (GOx) enzyme (which
acts as an oxidizing agent) is used as a biorecognition element,
where it oxidizes glucose to gluconic acid and transfers
electrons to oxygen, which is dissolved in the solution and then
converts into hydrogen peroxide, which can easily be detected
electrochemically. However, there are several examples where
direct electron transfer can take place from enzyme without
need of O2as an electron acceptor.
Generally, ultrathin
multilayer graphene nanosheets have been employed as a
transducing material for the biosensing of glucose.
There are
several reported examples where direct electron transfer occurs
from glucose oxidase. GO has also been used for the
recognition of glucose. Its biocompatibility with GOx led to
formation of a stable glucose sensor with a sensitivity of 8.045
mA cm2M1.
In another work, Niu and his co-workers
discovered a novel glucose biosensor based on direct electron
transfer process in graphene/ionic liquid/glucose oxidase
Their system showed a linear response up to 14
mM. Incorporation of CdS nanocrystal with graphene system
Figure 8. (a) Cyclic voltammograms of dierent components with graphene and without graphene. (b) Chronoamperometric response of
graphene/AuNPs/chitosan-modied electrode in N2-saturated phosphate buer on injecting the concentration of H2O2in 0.2 mM steps at working
potential of 0.2 V. The inset is amperometric response to H2O2concentration. Error bars = ±standard deviation. (c) Cyclic voltammograms at
graphene/AuNPs/GOD/chitosan-modied electrode in real blood sample and PBS mixing solutions containing 2.5, 3.5, 4.5, 5.5, 6.5, and 7.5 mM
glucose from down to up. Inset is the calibration curve corresponding to amperometric responses. Scan rate: 0.05 V s1. Error bar = ±standard
deviation. (d) Cyclic voltammetric measurements at graphene/AuNPs/chitosan-modied electrode in O2-saturated phosphate buer containing
various concentrations of glucose: 2, 4, 6, 8, 10, 12, 14, and 16 mM from bottom to top. The inset is the calibration curves corresponding to
amperometric responses at 0.2 and 0.5 V. Scan rate: 0.05 V s1. Error bars= ±standard deviation. Reproduced with permission from ref 136.
Copyright 2010 Elsevier.
ACS Biomaterials Science & Engineering Review
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provide very low detection limit of 0.7 mM with a linear
response between 2 to 16 mM.
Razmi and his co-workers
made a simple low cost electrochemical glucose biosensor
based on GOx-GQD|carbon ceramic electrode (CCE).
fabricate the biosensor at rst they simply coated the GQDs on
carbon ceramic electrode by drop casting method. The GQD-
modied carbon ceramic electrode (CCE) was activated and
coated with GOx to form the sensor. Their electrochemical
biosensor responds eciently between 5 and 1270 μM glucose
concentrations with the detection limit of 1.73 μM and
sensitivity of 0.085 mA cm2M1. Excellent performance of
the biosensor attributed to large surface to volume ratio,
excellent biocompatibility of GQDs, porosity of the GQD|
CCE, abundance of the hydrophilic edges as well as
hydrophobic surface in GQD which enhances the enzyme
absorption on the electrode surface.
There are also some other enzymatic detection methods
which have been reported based on graphene/Gold
nanoparticles(AuNPs)/chitosan composite.
As depicted in
Figure 8, the modied graphene/AuNP/chitosan showed
electrode good cyclic voltammograms response, chronoam-
perometric response and amperometric response with good
linearity. Not only glucose biosensor there are several other
biologically important molecules/macromolecules such as
nicotinamide adenine dinucleotide (NADH), DNA, hemoglo-
bin, cholesterol, catechol, hydrogen peroxide, ascorbic acid,
uric acid, dopamine, etc. which can also be measured by
graphene-based electrochemical biosensor. A brief detail of
these sensors has been described below.
NADH is a very essential coenzyme that participates in more
than 300 types of dehydrogenase enzymatic reactions.
Electrocatalytic oxidation of NADH has been studied as part of
the development of dehydrogenase-based biodevices. How-
ever, its electrooxidation at bare glassy carbon (GC) electrodes
in neutral solutions occurs at a high over potential (ca. 0.5 V)
because of electrode fouling and slow electron transfer
So the eective oxidation of NADH at low
potentials would lead to the path of development of NADH-
based biosensors. Li and his co-workers studied the electro-
chemical behavior of NADH on reduced graphene sheet lms.
They showed increased electron transfer kinetics and excellent
electrocatalytic activity compared with normal GC electrodes.
They observed that the oxidation of NADH occurs on bare
GC electrode at 0.75 V and the required voltage becomes
signicantly low to the 0.42 V using the reduced graphene
sheet lms/glassy carbon (rGSF/GC) electrode.
Zhang and
his group also prepared a NADH biosensor based on
noncovalent functionalized graphene with water-soluble
electro-active methylene green (MG).
The oxidation of
NADH at bare GC electrode occurs at +0.55 V. However, the
voltage changes to +0.40 V and +0.14 V for chemically reduced
graphene (CRG) and CRG functionalized with MG electrodes,
respectively. Shan et al. reported low potential electrochemical
detection of NADH as well as ethanol with the help of the