Graphene Quantum Dots: Syntheses, Properties, and Biological Applications$

TA Tabish and S Zhang, University of Exeter, Exeter, UK

r 2016 Elsevier Inc. All rights reserved.

1 Introduction 1
2 Approaches to Synthesis 2
2.1 Top-Down Methods 2
2.1.1 Acidic exfoliation and oxidation 2
2.1.2 Electrochemical methods 2
2.1.3 Solvothermal/hydrothermal methods 2
2.1.4 Other methods 2
2.2 Bottom-Up Methods 3
2.3 Surface Chemistry-Tuned Strategies 4
2.3.1 Surface functionalization 4
2.3.2 Doping with other elements 4
3 Properties 7
3.1 Structure 7
3.2 Luminescence Properties 7
3.3 Electrochemical and Electrocatalytic Properties 9
3.4 Toxicity and Biocompatibility 10
4 Biological Applications 12
4.1 Drug Delivery 12
4.2 Bioimaging 13
4.3 Biosensing 16
4.4 Antibacterial Applications 18
5 Summary and Outlook 18
References 18
Further Reading 21

1 Introduction

Nowadays, carbon based materials, particularly graphene and its derivatives, such as, graphene oxide (GO), reduced graphene
oxide (rGO) and graphene quantum dots (GQDs), have inspired intensive research efforts for several interdisciplinary sciences that
extend to a range of disciplines comprising chemistry, material sciences, physics, and nanotechnology [1–3]. Furthermore, gra-
phene and its derivatives have revolutionized scientific developments in biomedical applications such as drug delivery systems,
bioimaging, and anticancer therapy [4,5]. However the zero band gap of graphene limits its practical applications in optoelec-
tronics and photonics [6]. Surface modification, doping, as well as reducing the lateral dimensions of graphene into nanoribbon
and/or quantum dots (QDs) are considered the leading approaches to deal with its band gap phenomena [7]. As well as the
particularly improved quantum confinement effect (QCE), the edge effect will be prominent when graphene is transformed to
GQDs. These excellent characteristics provide GQDs with new physical properties for a variety of applications mentioned above.
GQDs are a class of zero-dimensional graphitic nanomaterials with lateral dimensions less than 100 nm in size and can be
found in single as well as multi layered form [1,8,9]. These GQDs are superior in chemical inertness, simplicity of production,
resistance to photobleaching, low cytotoxicity, and excellent biocompatibility in comparison to traditional semiconductor QDs,
hence making them applicable in sensors, bioimaging, optoelectronic devices, and so on. Moreover, the carboxyl and hydroxyl
groups at their edge empower them to exhibit good water solubility and also functionalization with numerous organic, inorganic
or biological classes. However, the concrete application of any nanomaterial in biology and medicine is distinguished and
investigated by its biocompatibility [10]. Though GQDs have been considered for several biological applications such as tissue
engineering, bioengineering, drug delivery, gene delivery, imaging and therapeutics but yet concerns associated with toxicity and
biosafety have become appropriate for its clinical trials.
In this article, we aim to review the recent developments of a protocol and a methodology for the preparation of GQDs and
their applications based on their excellent properties. We summarize the currently available approaches for the preparation of

☆
Change History: November 2015. T.A. Tabish and S. Zhang updated the Figure captions with the copy right permission of publisher.

Reference Module in Materials Science and Materials Engineering doi:10.1016/B978-0-12-803581-8.04133-3 1

2 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications

GQDs, and reveal the characteristics associated with their size, shape, doping, surface modification, and reaction to pH and
solvents. Subsequently, we describe the applications of GQDs in biological fields. Furthermore, we speculate on some critical
issues for further investigation and possible ongoing developments of GQDs.

2 Approaches to Synthesis

The synthesis of GQDs with excellent photoluminescence (PL) properties could be grouped into ‘bottom-up’ and ‘bottom down’
[11,12]. The bottom-up technique relies on the gradual chemical fusion of small aromatic molecules, this could be termed as
pyrolysis or carbonization. The bottom down technique is relatively the repeated reduction of carbon containing materials;
physical, chemical, or electrochemical routes could be used for this purpose. There may be another strategy surface chemistry
for the classification of fabrication techniques of GQDs. Surface chemistry-tuned strategies include surface functionalization and
doping other elements.

2.1 Top-Down Methods

2.1.1 Acidic exfoliation and oxidation
Treatments using conc. acids are common to exfoliate GQDs from carbon fibers [13,14], carbon nanotubes [15,16], GO [17],
graphite [18], coal [19,20], carbon black [21]; owing to their suitability for large scale productions but at the same time induce
negatively charged oxygenated groups, the resulting QDs become hydrophilic. Another problem is the abstraction of excess
oxidizing agents. A number of studies confirm the production of 1 m pitch based carbon fiber (Figure 1) [21] through acidic
exfoliation and oxidation and confirm their effectiveness.

2.1.2 Electrochemical methods

Electrochemical strategy is another widely used method to prepare GQDs. This approach is derived from the electrochemical
preparation of carbon dots and fluorescent graphene nanoribbons via defect-mediated fragmentation process. In 2011, Li and co-
workers firstly reported that green luminescence GQDs were synthesized in a 0.1 M phosphate buffer solution (PBS, pH¼ 6.86)
with a graphene film as the working electrode [22].
A novel electrochemical synthesis for GQDs on large scale in water was suggested by Deng et al., GQDs obtained with various
sizes and emission colors, exhibiting intrinsic peroxidase-like activity in glucose detection [23]. Very recently, the synthesis of size-
tunable GQDs, sizing 3, 5, and 8.2 ACHTUNGTRENUNG (0.3) nm electrochemically has been realized by using MWCNTs in a
solution of propylene carbonate [13]. Through this method, PL can also be tailored by changing the key process parameters as
required. Figure 2 is the schematic representation of two-step route for the electrochemical conversion of MWCNTs to GQDs.
Although electrochemical method is an effective way to fabricate GQDs derived from different starting materials where the
oxygen containing groups on the surfaces provide excellent aqueous solubility and the possibility of specific surface functiona-
lization, the drawback from this method is the complicated preparation process and low product yields which hinder its
development.

2.1.3 Solvothermal/hydrothermal methods

Hydrothermal synthesis typically uses thermally rGO sheets as the precursors, later treated with oxidizing agents to introduce
epoxy groups on the carbon lattice at the cleavage sites. A novel hydrothermal cutting mechanism for preoxidised graphene sheets
to ultrafine GQDs has been developed by Pan et al., and results in blue emission. The establishment of structural prototypes for
GQDs in acidic and alkali media is evident. It is proposed that the blue luminescence might have originated by the fluctuating sites
[11]. Figure 3 is the schematic representation of hydrothermal synthesis of GQDs explaining its mechanism, models of the GQDs
in acidic and alkali media and the electronic transitions of triple carbenes at zigzag sites [25].
Another approach established by Shen et al., is a surface passivated by polyethylene glycol (GQDs-PEG). They are manu-
factured by one spot hydrothermal reaction, utilizing small GO sheets with polyethylene as basic starting materials. These GQDs-
PEG with remarkable luminescence properties, double than the pure QGDs may offer a new type of fluorescence and upcon-
version material for biological applications [26].

2.1.4 Other methods

Another high production one spot GQD synthesis route is developed by Shin et al. [27] which uses microwave irradiation under
conditions of acidic and oxidizing nature. Using this technique GQDs can be manufactured from graphite by oxidative cleavage of
graphite and in situ rearomatization. These new GQDs having narrow distribution exhibit blue luminiscence.
Figure 4 shows the schematic representation of GQDs synthesis from graphite powder.
Lee et al. [28] described a fabrication technique allowing 10 and 20 nm GQDs from single layers of CVD developed graphene
using self-assembled BCP. The GQDs showed a hexagonal arrangement with a narrow size distribution.
 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications 3

Figure 1 (a) Schematic representation of oxidation cutting of CF into GQDs. (b) TEM images of GQDs (synthesized reaction temperature at
120 1C), inset of (b) is the HRTEM of GQDs. (c) AFM image of GQDs. (d) Size and height distribution of GQDs. (e) HRTEM image of the edge of
GQD, inset is the 2D FFT of the edge in (e). (f) Schematic illustration showing the orientation of the hexagonal graphene network and the relative
zigzag and armchair directions. (g) Representation of scheme of the edge termination of the HRTEM image in (e). (h) Proposed mechanism for the
chemical oxidation of CF into GQDs. Reproduced with permission [21]. Copyright 2012, Royal Society of Chemistry.

2.2 Bottom-Up Methods

Ruili et al. [29] developed multicolor PL GQDs with a uniform size of 60 mm using the BHC as a precursor. It was shown that
GQDs having a well-ordered morphology could be achieved by pyrolysis and exfoliation of large PAHs. It was further observed
that the sizing and properties of GQDs could be controlled by utilizing different aromatic molecules. The schematic process for
GQDs fabrication is clarified in Figure 5.
4 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications

Figure 2 Schematic representation of processing steps of photo luminescent GQDs from MWCNTs. Reproduced with permission [24]. Copyright
2012, Willey Online Library.

Recently, the carbonization of certain organic precursors like glucose and citric acid, presented strong potential to produce
GQDs. Tang and co-workers used glucose to prepare GQDs via microwave-assisted hydrothermal method [30]. Briefly, glucose was
firstly dehydrated to form C–C bonds, later the prevailing pressure conditions assisted the growth of the crystalline by the arranged
C–C bonds. Such manufacturing routes are described extensively. Heating the small organic molecules directly above their melting
point primes to condensation, nucleation, and successive development of GQDs. Organic salts (e.g., L-glutamic acid [31,32],
ascorbic acid [33], citric acid [34,35], cyclodehydrogenation of polyphenylene [30], and polyaromatic [36]) have been used
as precursors. These methods are simple, cost-effective, scalable, and tolerates ordinary inheritance of heteroatoms from the
precursors.

2.3 Surface Chemistry-Tuned Strategies

2.3.1 Surface functionalization
Kumar et al. [37] illustrated the examination of excitation dependent anomalous m–n type fingerprint PL transition in synthesized
amino functionalized GQDs (5–7 nm). The p–p band-to-band prompted transitions directed to excellent color production
subjected to variable excitation wavelength in the functionalized scheme. Ab initio outcomes proposed the establishment of an
interband condition similar to that of p orbital hybridization between C and N atoms at the boundaries. Another study reported
by Jiang et al. [38] also revealed that hydrogen peroxide and ammonia play a synergistic role on GO, in which hydrogen peroxide
slices the GO into reduced pieces and ammonia passivates the surface to give amine-modified GQDs. Similar study was
also conducted by Balanco et al. [39] with comparable results. Valizadeh et al. [40] have introduced carboxyl- and nitrite-
functionalized GQDs, (CNGQDs) as an active nitrosonium basis and effective catalyst for solvent reactions and fabrication of
azo-dyes in outstanding yields and with shorter reaction times. It has been illustrated in Figure 6.
Numerous chemical assemblies (e.g., diamine [41], thiol [41], hydrazide [42], and alkylamine [41,43]) have been examined
with GQDs. These electron providing classes frequently improve QY through radiative regrouping and frequently reason
noticeable wavelength alteration [44]. Such as, green oxygenated GQDs converted into blue after substituting carboxyl with
alkylamine [44].

2.3.2 Doping with other elements

Nitrogen doping is an influential approach to revise the characteristics of C based materials [45,46]. Doping GQDs with
heteroatoms delivers a striking mean of efficiently modifying their inherent properties and manipulating new mechanism
for innovative uses. Li et al. [47] reported an electrochemical method to glowing nitrogen-doped GQDs (N-GQDs) with
oxygen-based functional groups. Blue glow catalytic behavior was observed via N-GQDs having dissimilar N free positions with
 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications 5

Figure 3 Scheme of hydrothermal synthesis of GQDs (a) Mechanism for the hydrothermal cutting of oxidized GSs into GQDs: a mixed epoxy
chain composed of epoxy and carbonyl pair groups (left) is converted into a complete cut (right) under the hydrothermal treatment. (b) Models of
the GQDs in acidic (right) and alkali (left) media. The two models can be converted reversibly depending on pH. (c) The electronic transitions of
triple carbenes at zigzag sites. Reproduced with permission [25]. Copyright 2010, Willey Online Library.

Figure 4 Proposed scheme for the fabrication of GQDs from graphite powder by one-pot microwave irradiation under acidic conditions.
Reproduced with permission [27]. Copyright 2014, Willey Online Library.
6 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications

Figure 5 Processing illustration for the synthesis of GQDs by using HBC (1) as carbon source. Reproduced with permission [29]. Copyright 2011,
American Chemical Society.

Figure 6 Preparation of GQD-ONO from GQD-OH by using NaNO2. Reproduced with permission [40]. Copyright 2015, Elsevier.

atomic ratio of N/C 4.3%. The glowing property of N-GQDs makes it applications in bioimaging. Li et al. [48] reported
a solution chemistry technique to manufacture N-GQD. Figure 7 represents a solution chemistry approach to N-doped
colloidal GQDs.
Liu et al. [49] prepared N-GQD by a simple solvothermal technique utilizing dimethylforma-mide N source. Li et al. [50]
innovate a hydrothermal method for the making of N-GQDs by separating N-doped graphene. Hu et al. [51] showed another way
to synthesize it by using a hydrothermal treatment method. The maximum QY of 24.6% and high blue glow was attained. This
inexpensive and suitable development represents a prospective progression for large-scale fabrication.
 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications 7

Figure 7 Structures of N-Doped GQDs 1, 2 and an Undoped GQD 3 for comparison studies. Reproduced with permission [48]. Copyright 2012,
American Chemical Society.

3 Properties

3.1 Structure
Nevertheless GQDs can be deliberated as one brand of CDs [52]. CDs generally hold discrete structure. Usually, CDs are quasi
spherical nanoparticles involving of amorphous and crystalline fragments [53]. Many researchers ascertain the existence of crys-
talline sp2 carbon unit, these hold inferior crystallinity than GQDs [54,55]. In distinction, most of the GQDs are produced from
graphene, GO, and molecules with definite structure by way of benzene rings. Therefore, GQDs generally have graphene lattices
inside the dots, similar to the crystalline structure of single or multi-layered graphene. GQDs were primarily fabricated by Pan
et al., in 2010 [56], which tolerate a similarity to the crystalline structure of single or a few layered graphene. Most common
circular and elliptical shapes; triangular and hexagonal shapes have been produced also. Structural and physiochemical char-
acteristics of GQDs accounts to synthetic method, defects and functional groups.

3.2 Luminescence Properties

The investigation of optical properties have been started recurrently from the uprising of luminescent carbon based materials.
GQDs with various luminescent colors, ranging from UV to visible light and even near infrared region, have been produced with
several methods till today [56,57]. Though, optical parameters of GQDs are provocative subject, such as the luminescence
mechanism. For instance, the suggested QCE is not continuously perceived [58,59] also detailed and systematic mechanism of its
investigational technique is presently lack. However additional studies are desired to stimulus a vibrant depiction of the lumi-
nescence fundamentals and its phenomena, two ways of emission routes, band gap related intrinsic and surface related extrinsic
recombination courses, are extensively recognized since previous works [60].
GQDs frequently exhibit a well-established absorption peak around 230 nm falls in the UV region owing to the p–p
evolution in C¼ C within the graphene structure [58,59,61]. Some of the GQDs may show an absorption peak around the
280 to 360 nm range because of the n-p evolution of Carbon–Oxygen [62–68]. Due to QCE, the absorption peak positions
depend on the fabrication method and their size. For example, the absorption peak energy may fall by fluctuating the size of
hydrothermally prepared GQDs from 5 to 35 nm [69]. In addition, GQDs from carbon fibers and offered 1–4 nm to 7–11 nm
dependence (red shifting from 270 to 330 nm [70]. However, GQDs manufactured from glucose carbonization revealed
absorption peaks that were independent of the size [71]. However, the precise PL mechanism of GQDs remains disturbed
and needs additional investigations. Zhuo et al. observed that the upconversion PL property was not affected from the excitation of
the wavelength [60]. The UV–vis absorption spectrum of GQDs in water shows an absorption band at ca. 300 nm (Figure 8(a)).
Inset shows scattered GQDs brightened via light. A bathochromic shift of the excitation wavelength shifted the PL peaks to
longer wavelengths. However, an excitation independent behavior was observed when GQDs was prepared via ultrasonic reaction.
A strong peak at ca 407 nm and an invariable PL spectrum subjected to excitation wavelength between 240 and 340 nm.
Yang et al. has successfully computed and analyzed the quasi-particle energies and band gaps of GNRs using the first principle
Green's approach [72]. Supplement to the down-conversion photoluminescence properties, GQDs also show an excellent
up-conversion feature, Figure 8 shows the PL spectra by long wavelength light by up-converted emissions lated at ca. 407 nm,
which is similar to the down-conversion photoluminescence peak. This up-conversion property of GQDs could be well under-
stood incorporating the multi-photon active process similar to the previously mentioned CDs [73]. PL performance can be
influenced by several factors in addition to the size effect. Firstly, the photoluminescence property could be affected by the pH
value of the GQDs solution. Pan et al., reported that GQDs emitted strong PL under alkaline conditions. However, photo-
luminescence was almost entirely suppressed under acidic conditions. In addition, PL may be sensitive to the solvent selection
where the PL peak has been found to shift from 475 to 515 nm by varying the solvents (e.g., THF, acetone, DMF, and water).
Chhowalla et al. found that the PL property was changed with the reduction degree by hydrazine vapor [74]. For the applications
in biomedical and bioimaging arenas, upconversion fluorescence materials have recently attracted numerous interests. However,
the studies of upconversion photoluminescence (UCPL) in GQDs have been relatively scarce. Shen et al. firstly described that the
8 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications

Figure 8 (a) UV–vis absorption spectrum of the GQDs dispersed in water. Inset: optical photograph of the GQDs dispersed in water illuminated
under UV light (365 nm). (b) PL spectra of the GQDs at different excitation wavelengths. (c) Up-converted PL spectra of the GQDs at different
excitation wavelengths. Reproduced with permission [60]. Copyright 2012, American Chemical Society.

PEG passivated GQDs showed an upconversion emission peak ranging from 390 to 458 nm with the excitation wavelength varied
from 600 to 800 nm [75].
ECL behaviors of GQDs have only been investigated recently. The advantages of GQDs comprise steadier ECL and onset
potential closer to 0 V apparently due to faster electron passage by greater content of sp2 carbon. Li et al. [76] studied ECL
performances of the gGQDs and bGQDs in 0.05 M, pH 7.4 TBS. Figure 9(a) is representing the effect of potential cycle on the ECL
emissions. Furthermore, the cyclic voltammograms (CVs) in Figure 9(a) the peak was near to the ECL peak and therefore could be
allocated to the reduction peak of gGQDs.
 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications 9

Figure 9 (a) ECL‐potential curves and cyclic voltammograms (CVs) of the gGQDs (1,3) and background (2,4) with concentration of 20 ppm in
0.05 M Tris‐HCl (pH 7.4) buffer solution containing 0.1 M K2S2O8. Scan rate: 100 mV s1. (b) PL (lex ¼340 nm) and ECL spectra for the gGQDs‐
K2S2O8 system. (c) ECL‐potential curves of the background (1) and bGQDs (20 ppm) (2,3) in 0.05 M Tris‐HCl (pH 7.4) buffer solution containing
0.1 M K2S2O8. Reproduced with permission [76]. Copyright 2012, Willey Online Library.

3.3 Electrochemical and Electrocatalytic Properties

The electrochemical properties of the GQDS depend on arrangement of core and functional groups. Efficient transfer of electron
from GQDs is accelerated by the presence of large surface area and the plentiful edge sites [54]. Favaro et al. studied that GQDs
showed a vibrant reduction of the over potential as a function of the dopant conferring to the order: NBB4B, N. Furthermore,
they discovered that the oxidation state of a dopant is a measure of the selectivity of a particular reaction, owing to the fact that as-
prepared GOQDs obey the two-electron reduction path that would result in the formation of hydrogen peroxide, interestingly
reduction with NaBH4, the same would support a four electron reduction of oxygen to water [77]. Hetero-atom doping of GQDs
provides an interesting way of adjusting their intrinsic properties and exploring new dimensions for advanced device applications.
Li et al. [47] reported an electrochemical approach to luminescent and electro-catalytically active the N-GQDs with oxygen rich
functional group. These could be compared with commercially available Pt/C catalysts [78]. Similar electro-catalytic properties of
the N-GQDs for ORR has been investigated by Li and his team [63].
Catalytic activities of N-doped carbon materials could be further enhanced by understanding the size dependant catalytic
activities, therefore this area needs to be addressed.
10 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications

3.4 Toxicity and Biocompatibility

Even if toxicity and biosafety have not been comprehensively examined for GQDs. Although the applications of GQDs
may deliver unswerving developments and uprisings in the biomedical zone, but it could never be defined without
threat to human health; therefore, a profound investigation of toxicological and human protection studies are obligatory. It
is also precarious in order to expand a superior consideration of the derivation of toxicity. Recently, GQDs have been produced
in several means and broadly applied in bioimaging, drug delivery system, and therapy responses for anti-cancer activities,
but their toxicity has not yet been considered to the similar extent, particularly the toxicity mechanism. Table 1 summarizes
the in vivo and in vitro cytotoxicity effect of GQDs. Chong et al. [79] studied in vivo and in vitro toxicity of GQD showing
no noticeable effect on mice due to its smaller size. In vivo biodistribution experiment, it exposed no material accretion in
vital organs of mice. As Figure 10 shows, a lot of dark spots with tens of micrometers in diameter appear in the livers and spleens
from mice with GO-PEG, while nothing abnormal can be observed in the organs from mice with GQD-PEG. Wang et al. [80]
reported the toxic effects of GO and N-GQDs on red blood cells (RBCs). N-GQDs bothered the structure and conformation
of the lipid. So far, this is the earliest investigational study revealing the toxicity mechanism of N-GQDs in RBCs. Volarevic et al.
[81] investigated the in vivo toxicity of GQDs. Histopathological analysis demonstrated the accumulation in liver and reduced
Con A-mediated liver impairment. Roy et al. [82] reported that the toxicity of the GQDs with high biocompatibility values,
were defecated from the zebrafish's body without altering in any change its growth meaningfully. Su et al. [83] found low
toxicity of the PNF–GQD with cell viability assays. PNF–GQD hold the ability of targeting and imaging tumor cells and
presented much enhanced labeling efficacy compared with GQDs only. Chandra et al. [84] examined intracellular ROS produced
when HeLa cells are incubated with GQDs. As can be seen from Figure 11(b), P-GQDs showed lower intracellular ROS levels
compared to H-GQDs, which showed the highest levels of ROS. The cells incubated with H-GQDs showed highest green
fluorescence due to ROS formation. On the other hand, P-GQDs showed almost no fluorescence (Figure 11(c)). They indicate
the role of ROS production in the high toxicity exhibited by H-GQDs and also the ability of PEG matrix to mitigate this ROS-
caused toxicity.
Qin et al. [85] studied the mutual action of GQDs with macrophages and also the mechanism, this study showed that
they rarely altered cell viability and membrane integrity of macrophages. Furthermore, they showed that GQDs induce ROS
generation, apoptosis, autophagy, and inflammatory response via p38MAPK and NF-kB mediated signaling pathways in THP-1
activated macrophages. Zhang et al. studied the GQDs toxicity in stem cells and found out that it required more strict growth
conditions [65]. Shang et al. [86] observed the interactions of GQDs on human neural stem cells (hNSCs). They studied
the mechanism of GQD uptake by hNSCs and examined their effects on the proliferation and metabolic activity of hNSCs and
also found no substantial variation in the cell viability, proliferation, metabolic activity, and potential of stem cell. Lee et al. [87]
established the approach to realize effective and target specific delivery of GQDs using hyaluronic acid (GQDs-HA) as a
targeting agent.
The effective clinical understanding of GQDs needed a comprehensive approval/clearance from the living system within a
rational time period. The clearance level is ordered as intravenous4intramuscular4subcutaneous. All the above mentioned
studies propose the profound prospective of GQDs for in vivo and in vitro biological assessments, even though additional studies as
measurements of median lethal dose (LD50) parameters are also essential. Moreover, the biocompatibility and cytotoxicity of
GQDs also depends on the fabrication routes and parameters. Though the mechanisms involving the GQDs toxicity have not been
extensively explored. Several researchers have suggested that oxidative stress is one of the mechanisms responsible for the toxic
properties/effects of graphene related materials. The oxidative stress in target cells/molecules is produced by the generation of
reactive oxygen species (ROS). Antioxidant enzymes, such as superoxide dismutase or glutathione peroxidase, are capable to lessen
and abolish ROS. If homeostasis is not attained, cellular molecules, such as DNA, lipids, and proteins may be impaired. Cellular
molecules impairment by physical interaction with GQDs can be the additional promising mechanism of cytotoxicity. Toxicity
also relies on the electrochemical characteristics of GQDs, such as the functional groups, size, conductivity, nature of chemical
modifications and also the cell line and drug molecules. Functionalization/bio conjugation of the GQDs with organic molecules,
drugs, or targeting molecules significantly decreases the toxicity of GQDs as reported in several studies earlier. For instance,
PEGylated GQDs was effectively used for the anticancer drug delivery system. The popular of the in vivo studies of graphene related
materials are established on the assessment of vital tissue organs by histological and histopathological studies. It also deals the
biochemical parameters and complete blood count tests. But in the case of GQDs very few studies are available in the subject. It
may be further explored for GQDs in a variety of living systems. In vivo studies usually depend on the administration and
implantation/injection routes, low and high dose protocol, and the time period as well. It is evident from the current progress of
nanomedicine that in vivo and in vitro toxicity of GQDs is obligatory to fix its further practical applications. Recent studies show
that graphene-based materials could be utilized as a part of antimicrobial phenomena as a result of their flexibility. Direct
interaction of microscopic organisms with GQDs would instigate the loss of bacterial film and glutathione oxidation that the
amount GQDs antimicrobial activity adds to both layer interruption and oxidative stress. In addition to the biological toxicities,
environmental toxicities of GQDs applied in environmental remediation have not been investigated yet. Hence, this subject
demands further consideration.
 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications 11

Table 1 In vivo and in vitro cytotoxicity of GQDs

Materials/composites Study Concentrations /dose Model (cell line/ Highlights of the study Ref.
(with modifying agents) administration animal)

Carboxylated GQDs In vitro 50, 100, 250, and KB,MDA-MB231, No severe toxicity or [88]
500 mg/ml A549 cancer cells, morphological variations
and MDCK normal were observed
cell line
In vivo 5 and 10 mg/kg mice
GQD and GQD-PEG In vitro 10, 20, 40, 80, 160, WST-1, HeLa (or No obvious in vitro and [79]
320, and 640 mg/ml A549) in vivo toxicity, even
In vivo 20 mg/kg female Balb/c mice under multi-dosing
situation
GQDs In vitro 0, 25, 50, 100, 200, human neural stem No substantial variation [86]
200 mg/ml cells (hNSCs) was observed in the cell
viability
GQDs and HA-GQDs In vitro 0.1, 1, 2, 5 mg/ml MDCK and A549 Nontoxic behaviour [87]
In vivo 5 and 10 mg/kg Mice
CGQDs,H-GQDs, In vitro 0.5, 1, 2, 4, 8 mg/ml HeLa cells No significant toxicity was [84]
S-GQDs, and P-GQDs observed, PEGylation
process has made the
GQDs less cytotoxic as
compared to other
functionalized derivatives
GQDs In vitro 0–2 mg/ml HeLa, MCF-7 or Found high [82]
MCF-10A biocompatibility
In vivo 0–2 mg/ml Zebrafish (Danio
rerio).
GQDs In vitro 0, 1, 5, 10, 50, 100, THP-1 monocyte cell GQDs could induce [85]
200 mg/ml line inflammatory response,
apoptosis, and
autophagy in human
macrophages
GQDs In vitro 15, 30, 60, 90, 120, Hela cells Provides a potential for [89]
150, 180 360 mg/ml non-invasiveoptical
in vitro imaging
GQDs, pGQDs, cGQDs In vitro 10, 20, 50, 100, 200, Human lung Satisfactory biocompat- [90]
400 mg/ml adenocarcinoma ibility as fluorescent
A549 cells nanoprobes
GQDs In vitro 0, 25, 50, 100, human glioblastoma No toxic effects were [91]
200 mg/ml cell line U251 found
GQDs In vitro 0, 25, 50, 100, gastric cancer MGC- No toxicity as compared to [92]
200 mg/ml 803 cell and breast GO
cancer cell line MCF-
7
Insulin-conjugated GQD In vitro 0, 25, 50, 100, PC12 cells, T3-L1 No toxicity was observed [93]
200 mg/ml adipocytes
GQDs In vitro 0, 10, 20, 50, 100, MC3T3 cells Low cytotoxicity and [94]
200, 400 mg/ml MG-63 (Human excellent
osteosarcoma) cells biocompatibility
GQDs In vivo 0, 25, 50, 100, primary mouse No photo-toxicity was [95]
200 mg/ml þ 15 min splenocytes observed
photoirradiation
amine-functionalized In vitro 0, 10, 20, 50, 100, Hela cells Due to outstanding [96]
GQDs In vivo 200 and 500 mg/ml biocompatibility and
good antimycoplasma
properties
GQDs with various In vitro 0, 10, 25, 50, 100, A549 human lung GCDs did not diffuse in the [97]
functional groups (NH2, 200 mg/ml carcinoma cells and nucleus but were
COOH, and CO-N (CH3) human neural randomly dispersed in
2, respectively) glioma C6 cells cytolpasm, this showed
excellent
biocompatibility
12 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications

Figure 10 H and E stained tissue slices (liver, kidney, and spleen) of mice injected with GO-PEG (i.p.) and GQD-PEG (i.p. or i.v.) at the dose of
20 mg/kg per time. Mice were sacrificed at day 1 post seven injections in 14 days. While aggregated GO was found in the main organs (indicated
by golden circle) of mice injected with GO-PEG, no black dots were found in the groups of GQD-PEG. Bars indicated by white stripes in the image
are 50 mm. Reproduced with permission [79]. Copyright 2014, Elsevier.

4 Biological Applications

4.1 Drug Delivery

Graphene nanosheets are demonstrated to hold outstanding drug loading aptitude due to their specific surface area and interaction
with several molecules through p–p stacking, hydrophobic interfaces and physisorption. Having these qualities, GQDs are lesser to
tolerate excitedly cell approval and additionally compatible to reduce cytotoxic effects. These are predictable to be nontoxic,
harmless, operative and noticeable drug delivery routes [98]. Jing et al. [99] led one of the primary effort in this field. They
described a multifunctional capsule platform for fluorescence imaging. In their study the part of GQDs was luminescence imaging
as a tracer and marker for capsule targeting drug releasing and GQDs performed magnificently for imaging. Then, Chen et al. [100]
synthesized Graphene-HQDs (hybrid SiO2-coated quantum dots). This GQDs syndicates the benefits of graphene and HQDs and
exhibits excellent stability and fluorescence as well as enormously high drug loading efficacy. This directed to a sequences of
experiments by several groups for investigation of GQDs in drug delivery. Table 2 summarizes the variety of biological application
of GQDs. Wang et al. [101] demonstrated that GQDs have excellent capability in drug delivery and anticancer activity
improvement without any kind of functionalization. GQDs possess twofold role as an anticancer drug carrier and DNA cleavage
activity enhancer, which should be conceivably useful in cancer therapy. Generally, drug carrier vehicles intermingle with the cell
membranes and arrive into the cells by endocytosis. It is significant that the drug carrier restrains endosomal section and release
drug load in cytosolic sections. In this regard, functionalization has been successfully utilized to synthesize such drug vehicles that
could release drugs in the cytosol [102]. Lee et al. [87] have related the target particles to GQD. The fluorescence from the tumor
sustains that GQD was advent to the definitive end, while the strong fluorescence after in vitro imaging of CD44-overexpressed
A549 cells strongly provisions the achievement of the in vivo analysis. Nigam et al. [103] suggested a new approach that func-
tionalized HA-GQDs (hyaluronic acid) labeled human serum albumin for pancreatic cancer explicit drug delivery could be
exploited. Gemcitabine was encapsulated in albumin nanoparticles, and it is the ideal drug for pancreatic cancer cure. It was
 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications 13

Figure 11 (a) ROS production in solution containing samples with 20 mM H2O2 at 0 and 90 min. (b) Intracellular ROS in HeLa cells. (c) Intracellular
ROS imaged in HeLa cells. Green: DCF. Scale bar is 50 mm. Reproduced with permission [84]. Copyright 2014, American Chemical Society.

perceived that nano design meaningfully improved the bioavailability and continued drug release to pancreatic cancer cells. Sun
et al. reported that GQDs could be useful to drug delivery system jointly with specific drug by poor interface [104]. In addition, the
passivated PEG on GQDs also enhance drug loading capacity by hydrogen bonding and improve PL properties [105]. Figure 12 is
the schematic representation of drug delivery system through photodynamic therapy (PDT) flowing by bright-field image and red-
fluorescence image and also time dependent tumor growth after treatments.
Although these reports demonstrate that GQDs are an excellent candidate in drug delivery system as fluorescence label, more
efforts are needed to enhance the design and control of GQDs-based drug delivery systems.

4.2 Bioimaging
The tunable PL properties, good photostability, and low cytotoxicity also mark GQDs appropriate and auspicious in bioimaging.
Peng’s group cultured human breast cancer lines (cell T47D) with green luminescent GQDs [70]. GQDs presented a strong
excitation-dependent photoluminescence with high quantum yield (QY) and also delivered an excitation-dependent NIR
fluorescence emission. Even though the mechanism of NIR fluorescence emission was not entirely implicated. GQDs exposed
the potential to be used for in vitro and in vivo fluorescence imaging when used by a strong visible irradiation [64]. Apoptosis is
related to various irredeemable neurodegenerative, cardiac and cancer instigating diseases. Roy et al. reported a technique
exploiting the unique photoluminescent properties of plant leaf derivative GQDs modified with Annexin V antibody (AbA5)
to formulate (AbA5)-GQDs facilitating to mark apoptotic cells in live zebrafish also exhibited high biocompatibility [82]. A
lesser negative charge onto surface of GQDs can also play an important role in bioiamging. In this regard, Sun et al. [90]
investigated the durable PL of pGQDs than cGQDs in A549 cells. The less negative charges on pGQDs assurance them observe to
the negatively charged cell membrane more simply than cGQDs, thus accomplishing the operational uptaken by A549 cells and
this maintained the poorer cytotoxicity of pGQDs. Stem cell labeling was still a challenge due to the accuracy of stem cells.
However, Zhang et al. [106] used GQDs to incubate neurospheres cells (NSCs), pancreas progenitor cells (PPCs) and cardiac
progenitor cells (CPCs) and indicated that the cell morphology was discerned with the GQDs. Additionally, Zhu et al. [60] studied
the upconversion PL property of GQDs. This result exhibited that GQDs can be used as a successful optical probe under multi-
photon excitation.
 14
Graphene Quantum Dots: Syntheses, Properties, and Biological Applications
Table 2 Drug and gene delivery, tissue engineering and bioimaging applications of GQDs

Material Application Drug/gene cargo Highlights of the study References

GQDs DOX In vitro: A549 cells [120]

Results: the system could effectively prevent the leakage of drug molecules at neutral pH and
release them at acidic pH. The integration of luminescent GQDs, acid-cleavable acetal bond into
unique nanomaterials provide an excellent platform for bioimaging
GQDs DOX In vitro: human breast cancer cells MCF-7 and gastric cancer MGC-803 cells [101]
DNA cleavage Results: an outstanding ability in drug delivery and anti-cancer activity boost. The conjugates
enhanced DNA cleavage activity of DOX markedly
GQDs DNA cleavage, cleavage of apoptotic In vitro: human hepatocarcinoma cells HepG2 (ATCC HB-8065) [116]
markers and autophagy-related In vivo: BALB/c mice
(Atg) and genes delivery Results: GQDs alleviate immune-mediated fulminant hepatitis by interfering with T cell and
macrophage activation and possibly by exerting a direct hepatoprotective effect
GQDs DNA detection An effective fluorescent sensing platform for the detection of DNA based on Fluorescence [81]
resonance energy transfer (FRET), base pairing specificity of DNA and unique fluorescence
resonance energy transfer between the GQDs and GO to achieve quantitative analysis of DNA
GQDs DNA nanosensor This nanosensor can distinguish complementary and single-base-mismatched nucleic acid [117]
sequences with high sensitivity and good reproducibility. It showed a quite broad linear scope
and ultralow detection limit for DNA detection, and thus possesses excellent performance in
trace analysis
GQDs Imaging of apoptotic cells In vitro: human breast cancer cells (MCF-7) [82]
In vivo: adult male and female zebrafish
Results: this provided an opportunity to visualize the initiation and progression of apoptosis which
might help in understanding the apoptotic mechanism better
GQDs The determination of uric acid An analytical method for the determination of uric acid was developed based on its diminishing [112]
effect on the GQDs-Ce(IV) CL system. This has a good linearity, high sensitivity, and good
repeatability
GQDs bionanoconjugates Tumor biomarkers This technique enables clinically important biomarkers in buffer and serum. This offers an avenue [113]
for the detection of wide range of tumor biomarkers with enhanced sensitivity
Water soluble GQDs Ultrasensitive aptasensor for An anodic ECL was investigated from GQDs by using H2O2 as coreactant. The proposed ECL [114]
adenosine triphosphate detection aptamer sensor exhibited excellent analytical performance for adenosine triphosphate (ATP)
determination
GQDs Electrochemical biosensors GQD modified PG electrode coupled with probe ssDNA can be used as a platform to develop [111]
different kinds of electrochemical biosensors for the detection of various target molecules
GQDs Electrochemiluminescent DNA A signal-off ECL DNA biosensor was developed based on variation of the ECL intensity before and [115]
Biosensing after digestion of the DNA hybrid
GQDs Biosensor for trypsin On the basis of cytochrome c-induced self-assembled GQDs, a novel fluorescent biosensor for [110]
trypsin with remarkable fluorescence enhancement
Fluorinated GQDs and Long-term cellular imaging In vitro: HeLa cells [121]
nonfluorinated GQDs Results: GQDs-F was particularly suitable for long-term and real-time cellular imaging due to
ultrahigh photostability, in contrast to nonfluorinated GQDs
GQDs In vitro and in vivo fluorescence In vitro: MH-S cells [122]
imaging In vivo: mice
Results: GQDs could be used as a fluorescent labeling agent for bioimaging in both visible and NIR
regions
Si-GQDs Bioimaging In vitro: HePG2 cells [123]
Results: Si-GQDs not only serve as fluorescent chromophores in the composite material, but also
play a crucial role in the formation of mesoporous hollow silica spheres
GQDs Single bacterial spore for A unique bio-hydraulic cytobot confirms the intimate contact between the spore and pLGQDs in [124]
bio-electromechanical devices this example of graphene- based ‘biologically-actuating’ device. The device involves bio-
structure as an active component in electro-mechanics
Single layered GQDs Bioimaging In vitro: MCF-7 [7]
Results: The single-layer GQDs are demonstrated to be excellent probes for cellular imaging
GQDs Bioimaging In vitro: MG-63 [94]
Results: The GQDs have high stability and can be dissolved in most polar solvents owing to their
fine biocompatibility and low toxicity
GQDs Bioimaging In vitro: MC3T3 [125]
Results: researches in this article provide a direction to synthesize GQDs with multicolor

Graphene Quantum Dots: Syntheses, Properties, and Biological Applications

fluorescence through tuning surface chemistry, Up-conversion PL of GQDs is successfully
applied in near-IR excitation for bioimaging
Photo-excited GQDs Bioimaging In vitro: U251 [91]
Results: photo-excited GQDs displayed morphological and biochemical characteristics of both
apoptosis (phosphatidylserine externalization, caspase activation, DNA fragmentation) and
autophagy (formation of autophagic vesicles, LC3-I/LC3-II conversion, degradation of
autophagic target p62)
GQDs Bioimaging In vitro: NSCs, PPCs, CPCs (stem cell) [65]
Results: GQDs have demonstrated direct and easy penetration into stem cells without affecting
their viability, proliferation, or differentiation capacity. The GQDs in cells have shown strong
photoluminescence, good photostability, and low cytotoxicity
GQDs Bioimaging In vitro: MDA-MB231 [57]
Results: GQDs indicated that the near infrared GNPs can be used as noninvasive imaging agents.
The ex vivo images demonstrated the biodistribution profile and accumulation of
photoluminescent GNPs in organs
N-GQDs Bioimaging In vitro: HeLa [49]
Results: N-GQD can achieve a large imaging depth of 1800 mm, significantly extending the
fundamental two-photon imaging depth limit. It is nontoxic to living cells and exhibits super
photostability under repeated laser irradiation
N-GQDs Bioimaging In vitro: HeLa cells [51]
Results: Bright green luminescence is observed inside the cells, indicating that the N-GQDs have
been internalized by the HeLa cells and are mainly localized in the cytoplasm region and could be
used as efficient bioimaging probes

15
16 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications

Figure 12 (a) Bright-field image and (b) red-fluorescence image after subcutaneous injection of GQDs in different areas. The excitation
wavelength was 502–540 nm, and the collected fluorescence channel was 695–775 nm. (c) Photographs of mice after various treatments on the
1st, 9th, 17th, and 25th day. (PDT: GQDs þ light irradiation; C1: GQDs only; C2: light irradiation only.) (d) Time dependent tumor growth curves
(n¼ 5) after different treatments. po0.05 for each group. Reproduced with permission [87]. Copyright 2013, American Chemical Society.

4.3 Biosensing
The excellent properties of GQDs have facilitated their promising application in biological sensors. However, electrochemical
performances of the biosensor established the excellent utility of GQD in electrochemical biosensors. Higher stability and
precision of the biosensor in investigation of glucose and low toxicity of GQD for enzyme control are its significant benefits for its
practical application. A glucose-sensing system was designed based on electrostatic attraction between anionic fluorescent GQDs
and a cationic boronic acid-substituted bipyridinium salt (BBV) [107]. This presented that the electrostatic attraction between
GQDs and BBV ensued in the excited-state electron transfer from the GQDs to BBV and also reduced the fluorescence intensity of
the GQDs. A glucose biosensor using GQDs as enzyme-immobilization substrate-modified carbon ceramic electrode (CCE) was
also developed [108]. Razmi et al. [108] validated the excellent precision for the investigation of glucose, ascertaining the practical
application of glucose biosensor in clinical trials. It was also used for determination of blood sugar meditation in human plasma
models as well. Trypsin is the most significant gastric enzyme formed by the pancreatic acinar cells and it slices peptide bonds on
the C-terminal [109]. However, these approaches have particular shortcomings, such as the complication of conjugated poly-
electrolyte synthesis, fluorophore classification and photobleaching, callousness, also the requirement for various kinds of sub-
stances and the cytotoxicity of GQDs. Li et al. [110] studied GQDs for biosensing. The chemical reduction of GQDs to r-GQDs is
caused by trypsin cleaves peptide bonds of Cyt c into lysine, which also induce a subsequent increase in FL intensity. This is good
example of such kinds of sensors. Zhao et al. [111] have designed the GQDs modified PG electrode joined with probe ssDNA,
 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications 17

which can be used as a diverse kinds of electrochemical biosensor. Due to the excellent conductivity of graphene related materials,
the modified electrode can display adequate electrochemical response. Though, the probe ssDNA will prevent the electron transfer
between the electrochemical active species [Fe (CN)6]3/4 and the electrode. Thus several kinds of biosensors can be developed
with this scheme. Mohammad et al. [112] reported the CL of GQDs providing that Ce (IV) could directly oxidize GQDs to increase
CL emission and then they successfully applied to the determination of uric acid in humanoid plasma and urine samples. Shiddiky
et al. [113] developed an electrochemical immunosensor to reveal the epithelial cell adhesion molecule (EpCAM) antigen, which is
a common marker for tumors. The immunosensor may grow into an auspicious method for the primary recognition of tumor
biomarker in medical/living models. Lu et al. [114] proposed ECL aptamer sensor to measure ATP by relating ECL of GQDs and
aptamer method. Moreover, GQDs are demonstrated to be excellent ECL labeling agents, owing to excellent biocompatibility. The
fabrication of this ECL aptamer sensor based on GQDs ECL is represented in Figure 13.
Lou et al. [115] developed a biosensor for detection of DNA and it is based on site-specific cleavage of BamHI endonuclease
associated with the outstanding ECL properties of GQDs. A DNA biosensor was established based on difference of the ECL
intensity before and after incorporation of the DNA hybrid by using hepatitis C virus-1b genotype complementary DNA as a
model. Figure 14 represents its mechanism.
Though comprehensive work has been done to comprehend the phenomena by which enediynes cleave DNA, a continuous
assay for this mechanism is deficient. Biggins et al. [116] described the application of ‘molecular break lights’ to expand the
first continuous assay for cleavage of DNA by enediynes. Additionally, they directly linked the DNA cleavage by naturally
arising enediynes and as well as the restriction endonuclease BamHI. Zhou et al. [117] explored the applicability of GQDs
for DNA cleavage. GQDs and copper ions were stimulated by the statement that GO can be interpolated into DNA, and

Figure 13 Schematic representation of the ECL aptamer ATP sensor. (A) Immobilization of ssDNA1 on the surfaces of Au electrode. (B) Treated
with MCH to obtain a well-aligned DNA monolayer. (C) Hybridization of the two fragments in the presence of ATP. Reproduced with permission
[114]. Copyright 2013, Elsevier.

Figure 14 Schematic illustration of the ECL biosensor based on GQDs combined with endonuclease cleavage and bidentate chelation.
Reproduced with permission [115]. Copyright 2015, American Chemical Society.
18 Graphene Quantum Dots: Syntheses, Properties, and Biological Applications

investigational outcomes proved that GQD/Cu2 þ could cleave the DNA molecules more efficiently than GO/Cu2 þ with the
similar concentrations. The same study was conducted by Zheng et al. [118]. The investigation of the comprehensive cleavage
phenomena, optimization of cleavage circumstances, and documentation of the cleavage yields is needed to be investigated in
the future.

4.4 Antibacterial Applications

Synthesis of new antibacterial agents is becoming increasingly important in light of the emerging antibiotic resistance. Su et al. [26]
studied the antibacterial behavior of GQDs by using Escherichia coli (E. coli) as bacterial mode. The aptitude of GQDs treated
bacteria was examined by the ‘standard plate counting method.’ Furthermore, ROS level was sensed by the 3-(4,5-Dimethyl-
thiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) method [119]. Ristic et al. [95] reported that electrochemically produced
GQD generate ROS when photoexcited and killed two strains of pathogenic bacteria, methicillin-resistant Staphylococcus aureus and
Escherichia coli. Bacterial killing was monitored by the reduction in number of bacterial colonies in a standard plate count method,
the increase in propidium iodide uptake confirming the cell membrane damage, as well as by morphological defects visualized by
atomic force microscopy. The induction of oxidative stress in bacteria exposed to photoexcited GQD was confirmed by staining
with a redox-sensitive fluorochrome dihydrorhodamine. Mycoplasma is a simple infection mean in cell culture and clinical
models, and reasons pneumonia and further respirational syndromes. Jiang et al. found amine-functionalized GQDs, shows
good antimycoplasma properties. GQDs may submit new prospects for the progress of antimycoplasma agents. Another study for
Mycoplasma was conducted by Zhou and his team [97]. They reported that mycoplasma can sense to eukaryotic cells, excite cell
membrane damage through H2O2 discharge. Hydrogen peroxide can induce eukaryotic cells lipid peroxidation and speed up their
apoptosis.

5 Summary and Outlook

GQDs have gained huge interest in recent years because of their significant potentials for biomedical applications, owing to their
distinctive and tunable photoluminescence properties, remarkable physicochemical properties, high photostability, good bio-
compatibility, and small size. In this article, the latest developments are summarized in the synthesis, properties, cytotoxicity and
biological applications of GQDs. As presented above, GQDs, as a new graphene-based nanomaterial, have inspired intensive
research in environmental, biological and other fields because of their low cytotoxicity, excellent stability and resilience of PL
in vivo. There are many available methods for preparing GQDs, which have special properties, such as absorption, PL and
electroluminescence, which can be obtained by monitoring the band gap with unique size-tuning and chemical modification
approaches. Though some issues persist for additional research such as the mechanisms related to the optical properties of GQDs,
so a study of the detailed mechanism for PL in GQDs would be highly significant. The research on the preparation of high-quality
GQDs with uniform size and morphology is still ongoing; quantum confinement effect has not been illustrated in detail along
with its mechanism, so QCE and good quantum yield is yet to be addressed. Despite the diverse colored PL properties in the near
infrared region, the quantum yield of GQDs is still lower than 20%. This low percentage can limit the applications of GQDs.
Numerous favorable results related to their biocompatibility have been presented in recent years, which might open a new door
for their application in bioanalysis and may also provide improved designs for the applications in biotechnology such as cancer
diagnosis, drug delivery, and therapy. Therapeutic responses of GQDs have not been measured yet and as a result further studies
focusing on the chemotherapy, photodynamic therapy and combination of the two is required. There is no doubt that new
frontiers for antibacterial and neuroscience are also required to reduce neural and viral disorders in living systems.

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Further Reading

Ge, J., Lan, M., Zhou, B., et al., 2014. A graphene quantum dot photodynamic therapy agent with high singlet oxygen generation. Nature Communications 5, 4596.
Qian, Z.S., Shan, X.Y., Chai, L.J., et al., 2014. A universal fluorescence sensing strategy based on biocompatible graphene quantum dots and graphene oxide for the detection
of DNA. Nanoscale 6 (11), 5671–5674.
Some, S., Gwon, A.R., Hwang, E., et al., 2014. Cancer therapy using ultrahigh hydrophobic drug-loaded graphene derivatives. Scientific Reports 4, 6314.
Wang, X., Sun, X., Lao, J., et al., 2014. Multifunctional graphene quantum dots for simultaneous targeted cellular imaging and drug delivery. Colloids and Surfaces B:
Biointerfaces 122, 638–644.
Zhang, Y.Q., Ma, D.K., Zhuang, Y., et al., 2012. One-pot synthesis of N-doped carbon dots with tunable luminescence properties. Journal of Materials Chemistry 22 (33),
16714–16718.