REVIEW

published: 03 June 2020

doi: 10.3389/fchem.2020.00424

Recent Advances on Graphene

Quantum Dots for Bioimaging
Applications
Muhammad Rizwan Younis, Gang He, Jing Lin* and Peng Huang*
Marshall Laboratory of Biomedical Engineering, International Cancer Center, Laboratory of Evolutionary Theranostics (LET),
School of Biomedical Engineering, Shenzhen University Health Science Center, Shenzhen, China

Being a zero-dimensional (0D) nanomaterial of the carbon family, graphene quantum

dots (GQDs) showed promising biomedical applications owing to their ultra-small size,
non-toxicity, biocompatibility, excellent photo stability, tunable fluorescence, and water
solubility, etc., thus capturing a considerable attention in biomedical field. This review
summarizes the recent advances made in the research field of GQDs and place special
emphasis on their bioimaging applications. We briefly introduce the synthesis strategies
of GQDs, including top–down and bottom–up strategies. The bioimaging applications of
GQDs are also discussed in detail, including optical [fluorescence (FL)], two-photon FL,
Edited by:
magnetic resonance imaging (MRI), and dual-modal imaging. In the end, the challenges
Dong-Wook Han, and future prospects to advance the clinical bioimaging applications of GQDs have also
Pusan National University,
been addressed.
South Korea
Keywords: graphene quantum dots, synthesis method, bioimaging, fluorescence imaging, two-photon
Reviewed by:
fluorescence imaging
Min Zhou,
Zhejiang University, China
Suresh Kumar Kalangi,
Amity University Gurgaon, India INTRODUCTION
*Correspondence:
Jing Lin
Undoubtedly, the discovery of green fluorescence protein (GFP) and the development of organic
[email protected] fluorophores have fundamentally frameshifted the landscape of biomedical research, but their
Peng Huang limited photostability made long-term bioimaging a formidable challenge (Zheng et al., 2015).
[email protected] Although semiconductor quantum dots (QDs) have emerged as a potential alternative to the
developed organic fluorophores because of their photostability and brightness (Michalet et al., 2005;
Specialty section: Baker, 2010), the intrinsic toxicity, poor water solubility, and blinking characteristics restricted
This article was submitted to their widespread imaging applications (Zhao et al., 2018; Lu et al., 2019). Additionally, compared
Nanoscience,
to biological molecules, the larger-size semiconductor QDs (usually >500 kDa) might also affect
a section of the journal
the function and dynamics of target molecules (Zheng et al., 2015). Hence, the inherent limitations
Frontiers in Chemistry
of organic and inorganic QD-based fluorophores gave birth to the critical and constant efforts,
Received: 29 January 2020
exploring state-of-the-art fluorophores for bioimaging applications.
Accepted: 23 April 2020
Graphene quantum dots (GQDs), a latest zero-dimensional (0D) member of the carbon family,
Published: 03 June 2020
consist of single to few layers of graphene sheets with lateral dimensions of <10 nm (Li et al., 2013a;
Citation:
Benítez-Martínez and Valcárcel, 2015). Typically, GQDs not only possess the intriguing properties
Younis MR, He G, Lin J and Huang P
(2020) Recent Advances on Graphene
derived from two-dimensional (2D) graphene but also demonstrate extraordinary physicochemical
Quantum Dots for Bioimaging characteristics of the QDs, including edge effects, non-zero band gap, and quantum confinement
Applications. Front. Chem. 8:424. effects, by which they hold great potential in energy, electronic, and optical industry (Wang
doi: 10.3389/fchem.2020.00424 et al., 2016b; Chen et al., 2017a). In 2010, Pan et al. reported the successful synthesis of blue

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Younis et al. Recent Advances on Graphene Quantum Dots

luminescent GQDs via hydrothermal route by cutting graphene Top–Down Strategy

sheets and discovering their fluorescent properties for the first Chemical Exfoliation
time (Pan et al., 2010). Indeed, this work triggered innumerable Chemical exfoliation method involves the exfoliation of
experimental studies, and thus, a boom in GQD research has been precursor carbon materials such as graphene oxide (GO), carbon
witnessed to explore their potential bioimaging applications. nanotubes (CNTs), carbon fibers, etc., by strong oxidizing
Interestingly, owing to the excellent photostability, extended agents and acids. It is a facile, straightforward, and cheap
fluorescence, small size, biocompatibility, low cost, ease of synthesis approach for the mass production of high-quality
preparation, non-toxicity, and water dispersibility, fluorescent GQDs. Peng et al. prepared GQDs by exfoliating carbon fibers
GQDs surpass the conventional organic and semiconductor QD- with a mixture of strong sulfuric acid (H2 SO4 ) and nitric acid
based fluorophores, and emerged as a versatile and universal (HNO3 ) (Figure 2A). Owing to different stirring temperatures
fluorophore, offering unprecedented opportunities in bioimaging (80, 100, and 120◦ C), the resultant GQDs were in the size range
for precise diagnosis (Shen et al., 2012; Li et al., 2017; Zhang and of 1–11 nm with blue, green, and yellow emission, respectively,
Ding, 2018). Though physicochemical properties, fluorescence whereas the atomic force microscopy revealed the height of
mechanism, and applications of GQDs in energy, photocatalysis, GQDs at around 0.4–2 nm, suggesting single to few graphene
optoelectronic devices (Haque et al., 2018; Tian et al., 2018; layers. It is notable to mention that the chemical cleavage
Yan et al., 2019), sensing (Fan et al., 2015; Zhou et al., 2016; of the sp2 domain of carbon fiber actually determines the
Ozhukil Valappil et al., 2017), and cancer theranostics (Lin et al., successful formation of GQDs (Peng et al., 2012). Later, being
2016; Schroeder et al., 2016) have been well-reviewed, and the an abundant and the cheapest material, Ye et al. utilized coal
rapid advancements in bioimaging applications of GQDs strictly (anthracite, coke, and bituminous) as a precursor material to
demand a periodic update. Therefore, in this mini review, we fabricate GQDs (Figure 2B). Under acidic cleavage of coal,
attempt to spotlight the latest developments in GQD research, hexagonal GQDs within a size range of 3–6 nm were obtained.
focusing on their bioimaging applications. The different synthesis Interestingly, they suggested that the structure of coal possesses
strategies of GQDs, including top–down and bottom–up are crystalline carbon, which is highly suitable to undergo oxidative
briefly summarized, followed by a comparative and balanced displacement, leading to the formation of GQDs (Ye et al., 2013).
discussion on their bioimaging (fluorescence imaging, two- Subsequently, chemical exfoliation of graphite (Liu et al., 2013a)
photon imaging, magnetic resonance imaging, and dual-modal and asphaltene (Zhao et al., 2016a) was also reported to prepare
imaging) applications (Figure 1). Last, future perspectives to GQDs with excitation-dependent photoluminescence. Though
overcome the existing bottlenecks are also highlighted. We the combination of two strong acids (H2 SO4 and HNO3 )
envision that this review will offer a thorough understanding
about the great promise of GQDs in bioimaging, which may assist
in stimulating novel ideas, and hence, ultimately facilitate to push
the GQD research to a climax.

SYNTHESIS STRATEGIES OF GQDs

Though a number of well-established fabrication methods of
GQDs existed, the synthesis strategies are generally divided
into two main categories, including “top–down” and “bottom–
up.” In the top–down strategy, the bulk carbon materials
such as graphene, carbon black, etc., are usually cleaved
by chemical/electrochemical exfoliation, hydro/solvothermal
treatment, and microwave/ultrasonication, resulting in nanoscale
GQDs. Although the top–down strategy is highly suitable for
mass production because of the abundant precursor materials
and simple operation, the non-selective chemical cutting
leads to poor control over the size and morphology of the
ultimate product. Alternatively, the bottom–up strategy is
based on the gradual growth of small precursor molecules
(cyclic molecules, polymers) into nanosized GQDs by
carbonization, pyrolysis, chemical vapor deposition, etc.,
offering high controllability and fewer defects. However, the
poor solubility and aggregation of the resultant product is the FIGURE 1 | Graphene quantum dots (GQDs) for bioimaging applications. The
main limitation, which needs careful consideration. In the synthesis strategies of GQDs, including top–down and bottom–up are
following sub-sections, we will elaborate different synthesis summarized, followed by a comparative and balanced discussion on their
bioimaging (fluorescence imaging, two-photon imaging, magnetic resonance
methods underlying the umbrella of either top–down or imaging, and dual-modal imaging) applications.
bottom–up strategy.

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Younis et al. Recent Advances on Graphene Quantum Dots

effectively exfoliated the precursor materials, the removal of the fabrication of GQDs (Figure 2D). They suggested that the
excess sulfuric acid to purify the final product is a tedious size of the GQDs could be tuned by adjusting the concentration
process, which increases the overall synthesis cost. of both H2 O2 and W18 O49 . Notably, as the reactions products
Later, Dong et al. demonstrated that the concentrated HNO3 were only GQDs and water, the resultant GQDs could be directly
alone is sufficient to perform acidic cleavage of single-walled applied for bioimaging without any prior purification. Being
CNTs (SWCNTs), resulting in single to few layers of GQDs an acid-free strategy, mild oxidant-based exfoliation does not
(Figure 2C) (Dong et al., 2013). Following this, an acid vapor require extensive purification, offering simple, and environment
cutting approach was reported by Xu et al., using an extremely friendly fabrication of GQDs.
low volume (2 ml) of HNO3 to cleave metal–organic framework-
derived carbon, and the resultant GQDs were easily collected Electrochemical Exfoliation
via in situ filtration. As the direct contact between the precursor Electrochemical cleavage of carbon-based precursors, such
and the oxidizing agent is strictly avoided, this method offers as graphene paper, coke, graphite, and CNTs, is a potential
cheap and rapid synthesis of GQDs without any laborious strategy, which has been broadly employed to prepare single-
purification process (Xu et al., 2015). In another study, graphene layer GQDs with uniform size and high production yield.
oxide (GO) was exfoliated by HNO3 , following PEGylation and Depending upon the electrolyte, the electrochemical exfoliation
the reduction of exfoliated GO by hydrazine hydrate, yielding is generally divided into two sub-classes: water phase and organic
GQDs with an average diameter of 13.3 nm (Shen et al., 2011). phase electrochemical exfoliation (Zhou et al., 2016). By using
Owing to the surface passivation, GQDs exhibit high fluorescence organic electrolytes such as nitrogen-rich tetrabutylammonium
and up-conversion properties with a photoluminescence (PL) perchlorate and acetonitrile, Li et al. prepared nitrogen-doped
quantum yield (QY) of 7.4%. GQDs (N-GQDs) through an electrochemical cleavage of
Similarly, Kwon et al. reported the acidic cleavage of graphite graphene film at a cyclic voltammetry (CV) scan rate of 0.5
using HNO3 , followed by amidative cutting to fabricate GQDs, V/s (Li et al., 2012c). The resultant N-GQDs were single to few
exhibiting colorful PL (Kwon et al., 2014). They suggested that layers thick as suggested by the topographical height (1–2.5 nm),
the size of GQDs could easily be tuned by simply adjusting the around 2–5 nm in diameter, and possessed 4.3% atomic ratio
amine concentration. Instead of HNO3 , Maiti et al. employed of N/C. Owing to the heteroatom doping, N-GQDs displayed
perchloric acid to perform acidic exfoliation of GO to fabricate superior performance for electrocatalytic oxygen reduction
GQDs (Maiti et al., 2017). Notably, the prepared GQDs display reaction (OER) and high aqueous stability for long duration. In
strong excitation-independent PL with a QY of 14%, indicating a subsequent study, 3D porous graphene was electrochemically
the defect-free GQDs. On the other hand, spectroscopic analysis oxidized by acetonitrile and 1-butyl-3-methylimidazolium
revealed that the conjugated peripheral functional groups hexafluorophosphate (BMIMPF6 ) (Figure 3A), leading to the
with the planes of carbon backbone significantly contributed fabrication of single-layer, highly crystalline, and uniform blue
toward the excitation-independent PL property of the GQDs. fluorescent GQDs with a hydrodynamic diameter of 3 nm
Meanwhile, the longer PL life time (10 ns) as determined by the (Ananthanarayanan et al., 2014). Because of the hexagonal
correlated single-photon counting (TCSPC) conferred the great crystalline structure and the presence of BMIM+ on the surface
potential of GQD for biological probing. of GQDs, which contains electron-withdrawing N groups, these
Besides acid-based oxidizing agents, powerful oxidants GQDs presented excitation-dependent PL with a QY of 10%.
have also been reported for the chemical exfoliation/oxidation As the safe disposal of organic electrolytes is a serious
of carbon materials. For instance, Kundu’s group chemically environmental concern, water phase electrooxidation of carbon-
oxidized multiwalled CNTs (MWCNTs) using potassium based precursors showed great promise to fabricate GQDs.
permanganate (KMnO4 ) as an oxidant (Kundu et al., 2015a). For example, carbon fiber (CF) was electrochemically cleaved
The prepared GQDs were enriched with carboxyl (COOH) in borax (Na2 B4 O7 ·10H2 O) electrolyte to prepare bright
and hydroxyl (OH) functional groups, and possessed varying green fluorescent boron-doped GQDs (B-GQDs) (Fan et al.,
size ranges from 10 to 15 nm with an average hydrodynamic 2014). Another study demonstrated the formation of red
diameter of 12 nm. Instead of MWCNTs, Nair et al. oxidized GO fluorescent GQDs by electrochemically oxidizing graphite in
by KMnO4 to prepare high-quality GQDs with a fluorescence QY potassium persulfate (K2 S2 O8 , 0.01 M) (Tan et al., 2015). The
of 23.8% (Nair et al., 2017). They demonstrated one pot acid-free persulfate (S2 O2− −
8 ) generated radicals (SO4 ) performed the sharp
synthesis of GQDs with a production yield of >74%. Although electrochemical scissoring of the graphene sheets, leading to the
acid-based oxidizing agents and powerful oxidants showed formation of GQDs with an average hydrodynamic diameter of
high efficiency, the effective removal of these oxidizing agents 3 nm. Su et al. electrolyzed graphite rod in alkaline electrolyte
to purify the final product is a tedious and challenging task. (0.1 M NaOH), followed by reduction with hydrazine hydrate
Keeping this in mind, Shin et al. utilized non-acid mild oxidant (Figure 3B). The resultant GQDs exhibited excitation-dependent
such as oxone to exfoliate different naturally present carbon fluorescence emission and were two to three layers thick as
precursors (MWCNTs, graphite, charcoal, and carbon fiber), suggested by the atomic force microscopy (AFM) height profile
and the prepared GQDs exhibits blue fluorescence (Shin et al., (1–3 nm) and the dynamic light scattering (DLS) analysis (0.5–
2015). Subsequently, another mild oxidant hydrogen peroxide 2.5 nm) (Su et al., 2015). Later, an accelerated electrochemical
(H2 O2 ) was employed by Zhu et al. (2015a) to oxidize GO with exfoliation of the graphite rod was presented by Li et al. using
the assistance of tungsten oxide (W18 O49 ) nanowires, leading to ultraviolet (UV) irradiation and H2 O2 (Li et al., 2016c). They

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FIGURE 2 | Diagrammatical representation of the acidic exfoliation of different carbon-based precursor such as (A) carbon fiber (CF); reproduced from Peng et al.
(2012) with permission from the American Chemical Society. (B) Bituminous coal, reproduced from Ye et al. (2013) with permission from the Nature Publishing Group.
(C) Single-walled CNTs (SWCNTs), reproduced from (Dong et al., 2013) with permission from Elsevier. (D) GO; reproduced from Zhu et al. (2015a) with permission
from the Royal Society of Chemistry.

suggested that the electrons and the active radicals (HO· and H·) particular, they suggested that the strong electrolyte induced the
generated by UV irradiation and H2 O2 , effectively intercalated in rapid disintegration of graphite or graphene sheets, which leads
the graphite working electrode and broke the C–C bond, thereby to a low product yield, whereas the prolonged electrochemical
favoring the rapid generation of small-size GQDs with high yield. cleavage and the suppression of intercalation by weak electrolytes
Instead of single graphite rod and electrolysis, Bahadur’s result in an effective and steady fabrication of GQDs (Figure 3D).
group prepared GQDs by thermally treating two graphite Impressively, this strategy could be further extended to other
rods prior to electrochemical oxidation within an electrolyte weak electrolytes, e.g., hydrogen fluoride (HF) and hydrogen
containing both NaOH and citric acid (Ahirwar et al., 2017). The sulfide (H2 S), for the electrochemical cleavage of other carbon
thermal treatment at 1,050◦ C induced several surface defects on precursors, including CNTs, CF, graphite, and graphene.
graphite rods, which provided greater number of active sites to
facilitate the electrochemical oxidation reaction (Figure 3C). The Hydrothermal/Solvothermal Exfoliation
as-fabricated GQDs were around 2–3 nm in diameter and showed Compared to other synthetic processes, hydro/solvothermal
excitation-dependent PL property. Recently, weak electrolyte exfoliation is a simplified approach to prepare GQDs. For the
(ammonia solution, NH4 OH)-mediated electrolysis was reported first time, Pan et al. fabricated blue luminescent GQDs by
by Huang et al. for the first time (Huang et al., 2018). In the hydrothermal exfoliation of GO sheets (Pan et al., 2010).
their study, a graphene paper as a carbon precursor, completely Prior to the thermal treatment, GO sheets were chemically
oxidized within 120 min of electrochemical reaction, yielded oxidized, and thus, the resultant GO carbon lattice possessed
highly crystalline GQDs with a product yield of 28%. The several epoxy groups. Interestingly, these epoxy groups acted
rapid electrochemical cutting is attributed to the low ionization as a cleavage points and were completely broken during
capacity of the weak electrolyte, which effectively suppressed hydrothermal reaction to yield GQDs. Meanwhile, the simple and
the intercalation. Second, due to the high concentration of the highly efficient approach (approximately 35 wt% conversion rate)
•OH radical, the weak electrolyte speeded up the electrochemical to fabricate GQDs by hydrothermal treatment with the aid of
cutting and increased the overall reaction efficiency. During the potassium superoxide (KO2 ) was demonstrated by Zhao et al.
electrochemical cleavage, the electrolyte solution rapidly turned (2017). The as-prepared water-soluble GQDs exhibited yellow
from colorless to black due to the rapid formation of GQDs. In emission with a photoluminescence QY of 8.9% (Figure 4A).

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FIGURE 3 | Illustration of GQD fabrication by electrochemical approach using 3D porous graphene (A); reproduced from Ananthanarayanan et al. (2014), with
permission from Wiley. (B,C) Graphite rods; reproduced from Su et al. (2015) and Ahirwar et al. (2017) with permission from Wiley and American Chemical Society. (D)
Graphene paper; reproduced from Huang et al. (2018) with permission from the American Chemical Society.

Instead of chemical oxidation, GO sheets were fragmented by traditional carbon precursors for top–down GQD formation,
pulsed laser irradiation, followed by hydrothermal treatment to they employed durian fruit as a precursor for the fabrication of
fabricate fluorescent GQDs with tunable emission (Qin et al., S-GQDs. The fruit was first smashed and then hydrothermally
2015a). Compared to chemical exfoliation, pulse laser-mediated treated at 150◦ C for 12 h in the presence of a platinum (Pt)
ablation is a versatile and clean approach to prepare high-quality sheet as a catalyst. The structure of the resultant S-GQDs
GQDs. To avoid the entire pre-processing step, Chen et al. were highly crystalline, while their size ranged from 2 to
reported on one pot hydrothermal exfoliation of starch to prepare 6 nm with an average hydrodynamic diameter of 4 nm. The
GQDs (Chen et al., 2018). Owing to the absence of oxidizing topographical height profile (0.5–1.0 nm) revealed single- to few-
agents, the extensive post-processing to purify the final product layer-thick S-GQDs. Owing to the S doping, S-GQDs displayed
was avoided. Second, as the final reaction products were only remarkable PL performance with a much higher PL quantum
GQDs and water (Figure 4B), the synthesis process is purely yield (79%) than most of the reported GQDs and extraordinary
green and eco-friendly. Furthermore, the entire hydrothermal photo/chemical stability. Following the hydrothermal approach,
reaction was completed within 120 min, which suggested the N,S-co-doped GQDs (N,S-GQDs) were recently developed by
rapid hydrothermal cutting of the precursor. Kulchat’s group using citric acid as a source of carbon and
Alternatively, some researchers utilized oxidants or mild cysteamine hydrochloride for nitrogen and sulfur, respectively
oxidants to accelerate the overall hydrothermal reaction. For (Boonta et al., 2020). The resultant N,S-co-doped GQD possessed
instance, Halder et al. prepared GQDs by one pot hydrothermal an average hydrodynamic size of 3.0 nm with a size distribution
exfoliation of GO sheets in the presence of H2 O2 (Halder of 1.1–5.4 nm. Though an excitation-dependent PL was observed,
et al., 2018). Notably, H2 O2 effectively scissors the GO sheets the co-doped GQDs were used for the fluorescence sensing of
during thermal treatment and thus significantly accelerated cobalt ions by metal–ligand interaction mechanism as cobalt
the exfoliation reaction. The obtained GQDs were 5 nm in ions interacted with carbon and the surface nitrogen and sulfur
diameter, exhibited high photostability and non-cytotoxicity. functional groups.
Similarly, ascorbic acid-assisted hydrothermal cutting of graphite Apart from the hydrothermal fabrication, Zhu et al. reported
to synthesize GQDs was also demonstrated (Cirone et al., 2019). the solvothermal approach to produce GQDs (Zhu et al., 2011).
Recently, in contrast to the GQDs, Wang et al. prepared sulfur- Green fluorescent GQDs with 11% fluorescence QY were formed
doped GQDs (S-GQDs) by one pot hydrothermal exfoliation by the solvothermal exfoliation of the GO sheets in dimethyl
reaction (Wang et al., 2018a). Meanwhile, compared to the formamide (DMF) solvent for 5 h at 200◦ C. The single- or

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FIGURE 4 | (A) Fabrication of GQDs by the hydrothermal exfoliation of GO with the assistance of potassium superoxide (KO2 ); reproduced from Zhao et al. (2017)
with permission from Elsevier. (B) Hydrothermal cutting of starch to synthesize GQDs; reproduced from Chen et al. (2018) with permission from the Royal Society of
Chemistry. (C,D) Acid-free solvothermal exfoliation of different carbon-based precursors to fabricate GQDs; reproduced from Shin et al. (2015) and Tian et al. (2016)
with permission from the Royal Society of Chemistry and Elsevier.

bilayer-thick GQDs as suggested by their height (1.2 nm) were Md Noor et al., 2018). At a different reaction time (0.5–
about 5.3 nm in diameter. Following this, Wang’s group also 8 h), pN-GQDs, with a size range from 2.8 to 6.3 nm, were
fabricated green fluorescent GQDs in DMF, but the obtained formed, while the concentration of pyrrolic N determined
GQDs were a little bigger (4.92 nm) in size (Yu et al., 2017). the luminescence properties of the resultant pN-GQDs as the
Similar to oxidant-assisted hydrothermal cleavage, an oxone- brightness of the emitted white light increased with an increase in
assisted solvothermal exfoliation of different carbon precursors, the concentration of pN-GQDs. Importantly, the concentration-
including charcoal, MWCNTs, graphite, and CF (Figure 4C) dependent enhanced brightness feature is advantageous for the
was demonstrated by Shin et al. (2015). Later, Tian et al. fabrication of white light-emitting diodes (LEDs) as well as for
presented mild oxidant (H2 O2 )-assisted solvothermal exfoliation lighting applications.
of graphite (Figure 4D). Impressively, the non-acid oxidant-
assisted solvothermal reaction do not require extensive dialysis Microwave/Ultrasound-Assisted Exfoliation
for purification and thus exhibited facile, environment friendly, Being dependent on the conventional heating sources (oil bath,
and low-cost fabrication of GQDs (Tian et al., 2016). During electric oven), the chemical/electrochemical and hydrothermal
solvothermal reaction, the tunable PL of GQDs was studied by Qi exfoliation methods usually suffer from long reaction time and,
et al. Following different reaction conditions, two different-sized thus, are not suitable for the large-scale industrial production
GQDs (2.6 and 4.5 nm) with different surface chemistry were of GQDs. In contrast, microwave irradiation can remarkably
prepared (Qi et al., 2018). They suggested that the PL emission shorten the reaction time by providing uniform heat, allowing
is largely influenced by the particle size and surface oxidation the rapid formation of high-quality GQDs. Hence, the integration
as both the larger-size particles and higher surface oxidation of microwave irradiation with other exfoliation approaches is
lead to bathochromic shift in the PL emission. Recently, Noor an effective strategy to achieve a high yield of GQDs in less
et al. performed solvothermal reaction in DMF and prepared time. Li et al. for the first time reported on the preparation of
white light-emitted pyrrolic N-doped GQDs (pN-GQDs) (Farain GQDs by microwave-assisted chemical cleavage of GO sheets

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under acidic conditions (Li et al., 2012a). The acidic cleavage utilization of microwave-assisted strategy and an IL, which
induced several epoxy groups on the GO sheets, which were easily contained heteroatoms.
ruptured under microwave treatment, leading to the formation In addition to microwave-assisted exfoliation, ultrasound-
of green fluorescent GQDs (Figure 5A). The height profile assisted exfoliation have also been employed for the facile
indicated single-layer GQDs with an average of 4.5 nm diameter. and mild fabrication of GQDs. In brief, ultrasound produces
Interestingly, they further suggested that the blue fluorescent alternating high/low pressure waves in a liquid, which lead to
GQDs can be designed by the reduction of surface functional the constant formation and abrupt collapse of small vacuum
groups of green GQDs with sodium borohydride (NaBH4 ). By bubbles. Finally, these cavitations generated high-speed liquid
triggering the acidic cleavage and reduction simultaneously, jets, deagglomeration, and strong hydrodynamic shear forces (Li
microwave irradiation combined oxidation and reduction steps et al., 2011). Taking an advantage of these features, ultrasound is
into a simple one step process toward GQD fabrication. Luo et al. a favorable strategy to fracture the layered structures of carbon-
reported the two-step fabrication of white fluorescent GQDs (Luo based precursors into GQDs. In 2012; Zhuo et al., for the
et al., 2016). First, graphite was transformed into yellowish green first time, prepared GQDs from the facile and direct ultrasonic
fluorescent GQDs under microwave-assisted acidic cleavage, exfoliation of graphene (Zhuo et al., 2012). Since then, a range
followed by the microwave assisted hydrothermal (MAH) of carbon materials such as cheap graphite, MWCNTs, CF,
treatment of GQDs to yield white fluorescent GQDs. Apart from and GO have been explored for the ultrasonic preparation of
the chemical oxidation, a simple microwave exfoliation of citric GQDs in either organic solvent or aqueous solution (Zhou
acid (a common food additive) at 700 W for only 4 min was et al., 2016). Using graphite as a precursor, Song et al. reported
demonstrated to prepare GQDs as shown in Figure 5B (Zhuang the ultrasonic synthesis of GQDs (Song et al., 2014). In a
et al., 2016). During the reaction, the solution turned from typical experiment, graphite intercalation compounds (GICs)
colorless to yellow depending upon the reaction time. Similarly, a were first prepared by mixing potassium sodium tartrate and
time-dependent fluorescence emission was also noticed as a slight graphite in a mass ratio of 10:1, followed by hydrothermal
increase in the reaction time shifted the emission peak from 411 treatment for 24 h at 250◦ C (Figure 5E). The aqueous dispersion
to 466 nm. Following this, the rapid preparation (3 min) of white of the as-synthesized GICs was further sonicated for an hour
fluorescent GQDs using trinitropyrene was also recently reported to produce GQDs. The resultant GQDs were about 1–5 nm in
by Wu’s group (Li et al., 2018c). diameter and 0.5- to 1.5-nm thick as indicated by the height
Later, different researchers employed the microwave-assisted profile. Subsequently, Hassan et al. presented the direct ultrasonic
method to prepare heteroatom-doped GQDs. Instead of exfoliation of few-layer graphene sheets (FLGs) and activated
microwave-assisted chemical cleavage, Sun et al. prepared graphene to prepare GQDs (Hassan et al., 2014). The FLGs were
fluorine-doped GQDs (F-GQDs) by the microwave-assisted transformed into activated graphene by treating with potassium
hydrothermal (MAH) exfoliation of fluorinated GO (FGO) sheets hydroxide at a high temperature, followed by ultrasonication
(Sun et al., 2015). First, the FGO sheets were chemically cleaved in for 2 h to yield 16.3 wt% activated GQDs (aGQDs), while the
a mixture of strong oxidizing agents (HNO3 /H2 SO4 ), followed by yield of the GQDs produced by the direct sonication of FLGs
microwave treatment at 650 W for 6 h. Similarly, the formation of was 3.4%. Following this, Zhang et al. recently fabricated blue
F-GQDs using glucose as a precursor was demonstrated by Yang’s luminescent GQDs from the one pot ultrasonic exfoliation of
group (Yousaf et al., 2017). The as-obtained F-GQDs were highly coal (anthracite) in DMF (Figure 5F). The as-obtained GQDs
crystalline and smaller in size (2.38 ± 0.04 nm) than GQDs. were highly stable, around 3.2 nm in diameter, and showed
Moreover, compared to GQDs, F-GQDs revealed green shift two fluorescence emission modes due to the surface defects
in PL, which is attributed to the fluorine doping. An acid-free and sp2 domain of carbon (Zhang et al., 2019b). Interestingly,
doping of GQDs were reported by Hai et al. (2015). In their work, ultrasonication approach also facilitated the fabrication of
graphite and borax were used as a carbon and boron source, heteroatom-doped GQDs. For example, Zhao’s group employed
respectively, for the one-step microwave irradiation-mediated the chlorinated CF as a precursor for the ultrasonic formation
synthesis of boron-doped GQDs (B-GQDs) (Figure 5C). The as- of chlorine-doped GQDs (Cl-GQDs) in N-methylpyrrolidone
prepared B-GQDs exhibited excitation-independent PL with a (Zhao et al., 2015). In their work, hydrochloric acid (HCl)
21.1% QY. induced the chlorination of CF as well as cut the CF into
Recently, Ren et al. prepared N-doped GQDs with a small pieces. The resultant Cl-doped CF was then sonicated
hydrodynamic size of 5.6 nm and a QY of 8% using both in N-methylpyrrolidone for 10 h to produce Cl-GQDs. As
triethanolamine and sodium citrate as a precursor, respectively the heteroatom doping alters the physicochemical properties
(Figure 5D) (Ren et al., 2019). In contrast to single-atom doping, of the GQDs, the Cl-GQDs exhibited substantially improved
Kundu et al. presented a simple strategy to develop multiatom photovoltaic performance.
(S, F, and N)-doped GQDs (Kundu et al., 2015b). In their
study, MWCNTs as a carbon precursor were first dispersed in Bottom–Up Strategy
an ionic liquid (IL) via ultrasonication, followed by microwave Carbonization/Pyrolysis
treatment for 15 min at 1,100 W. After purification via dialysis The carbonization of small molecules/organic-based precursors
and filtration, the multiatom-doped GQDs of around 2-nm size is a simple and straightforward approach to fabricate GQDs,
were obtained with high yield (78%). The rapid and efficient and thus have been widely explored in recent years. Specifically,
formation of heteroatom-doped GQDs was ascribed to the the small organic-based precursor molecules are heated at a

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Younis et al. Recent Advances on Graphene Quantum Dots

FIGURE 5 | (A) Illustration of the preparation of GQDs by microwave-assisted chemical cleavage of GO sheets; reproduced from Li et al. (2012a) with permission from
Wiley. (B) Microwave exfoliation of citric acid to fabricate GQDs; reproduced from Zhuang et al. (2016) with permission from Wiley. (C) Preparation of boron-doped
GQDs (B-GQDs); reproduced from Hai et al. (2015) with permission from the Royal Society of Chemistry. (D) Preparation of nitrogen-doped GQDs (N-GQDs);
reproduced from Ren et al. (2019) with permission from the American Chemical Society. (E) Ultrasonic preparation of GQDs using graphite; reproduced from Song
et al. (2014) with permission from Wiley, and (F) coal as a precursor; reproduced from Zhang et al. (2019b) with permission from the American Chemical Society.

temperature higher than their melting point, which triggered nitrogen-doped GQDs (N-GQDs) via direct carbonization using
the nucleation, condensation, and the subsequent fabrication ammonium citrate as a precursor for both carbon and nitrogen,
of GQDs. A number of precursors, including organic salts, respectively (Yin et al., 2016). The homogenous aqueous solution
ethanolamine, acetylacetone, amino acids, co-factors (ascorbic of ammonium citrate was carbonized at high temperature
acid), humic acid, coffee grounds, carbohydrates (sucrose or (200◦ C), which led to the production of GQDs (Figure 6A).
glucose), citric acid, etc., have been used for the bottom–up Following the similar concept, organic precursors were also
preparation of GQDs (Zhou et al., 2016). It is noteworthy carbonized to fabricate heteroatom-co-doped GQDs. In 2015,
to mention that the preparation of different types of GQDs, Qu’s group fabricated nitrogen, sulfur-co-doped GQDs (N,S-
e.g., GQDs, heteroatom-doped GQDs, and heteroatom co-doped GQDs) using thiourea and citric acid, respectively, as a precursor
GQDs largely depends on the chosen precursor. For instance, (Qu et al., 2015). Thiourea provides S and N atoms, while
HCl and fructose were chosen as a source of Cl and carbon citric acid was used as a source of carbon. In their experiment,
to prepare Cl-GQDs (Li et al., 2013b). Under hydrothermal both the precursors were first efficiently dissolved in DMF,
treatment, the O and H groups present within fructose were followed by thermal treatment at 180◦ C in a heating mantle. The
dehydrated, while the carbon formed the nucleus of GQDs. purified GQDs were 1-nm thick, 4.5 in diameter, and exhibited
In the meantime, HCl catalyzed the reaction and offered a Cl excitation-dependent emission owing to the co-doping of N and
dopant. Recently, Wang et al. synthesized highly fluorescent S. Following the carbonization approach, the development of

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Younis et al. Recent Advances on Graphene Quantum Dots

FIGURE 6 | (A) Illustration of the carbonization process for GQD formation using ammonium citrate as a precursor; reproduced from Yin et al. (2016) with permission
from Elsevier. (B) Pyrolysis of glucose; reproduced from Tang et al. (2014) with permission from the American Chemical Society. (C) Pyrolysis of
hexa-peri-hexabenzocoronene (HBC) for GQDs; reproduced from Liu et al. (2011) with permission from the American Chemical Society. (D) Fabrication of N-GQDs
using 1,3,5-triamino-2,4,6-trinitrobenzene (TATB) precursor; reproduced from Li et al. (2016b) with permission from Wiley.

other heteroatom-doped GQDs such as P-GQDs, Si-GQDs, etc., (800–1,200◦ C) and pressure (4.0 GPa) (Zhu et al., 2015b).
is highly expected in the future. The resultant N-GQDs showed tunable band gap, possessed
For the pyrolysis-mediated fabrication of GQDs, the precursor graphitic nature, and N-doping in the carbon lattice without
molecules are generally divided into two categories: aromatic any surface functional groups, which induced a negative effect
(hexa-peri-hexabenzocoronene, HBC) and non-aromatic (citric on the electronic cloud (π electrons) of GQDs. Similarly, the
acid, glucose, etc.). Though the aromatic molecules possessed formation of N-GQDs using citric acid and melamine as a
a pi system, the non-aromatic precursor molecules need intra- carbon and N source, respectively, was also reported (Zhou
and intermolecular dehydrogenation (Ozhukil Valappil et al., et al., 2017). In another study, green fluorescent N-GQDs were
2017). Recently, Lee et al. fabricated single crystalline GQDs formed using 1,3,5-triamino-2,4,6-trinitrobenzene (TATB) as an
using D-glucose as a precursor (Lee et al., 2019). The as- aromatic precursor (Figure 6D). It has been noticed that the
prepared GQDs exhibited blue fluorescence, possessed hexagonal intramolecular carbonization of TATB during pyrolysis plays
crystalline structure, and were 5 nm in lateral dimension. In a dominant role in the fabrication of N-GQDs (Li et al.,
contrast, using glucose as a precursor, N-GQDs were synthesized 2016b). In contrast to high temperature/pressure carbonization,
by Tang et al. in the presence of ammonia (NH3 ) (Tang et al., Habiba et al. transformed the benzene into GQDs by pulsed
2014). They suggested that NH3 not only doped N in situ laser irradiation (Habiba et al., 2015). They suggested that
but also performed the inter/intra-molecular dehydrogenation under pulsed laser irradiation, the aromatic molecules generated
of glucose (Figure 6B). Owing to the resemblance of graphene free radicals, which first reorganized into carbon nanospheres
fragments with the HBC and other polyaromatic hydrocarbons and then combined together to yield GQDs. Meanwhile, the
(PAH), the production of GQDs have also been demonstrated longer laser treatment yields bigger-size GQDs. Recently, a facile
by the controlled pyrolysis of PAH-based precursor molecules approach to design highly pure GQDs in the absence of any toxic
as shown in Figure 6C (Liu et al., 2011). Besides PAH, a chemical was presented by Lee et al. (2020). Briefly, they annealed
nitrogenous aromatic precursor such as melamine has been used SiC followed by etching with low vacuum H2 gas, resulting in
by Xie’s group to synthesize N-GQDs under high temperature highly crystalline and pure GQDs with fewer defects and the

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Younis et al. Recent Advances on Graphene Quantum Dots

lack of surface functional groups of oxygen. Importantly, the annealing of C60 at 500–550 K triggered the adatom-vacancy
regulation of vacuum pressure altered the morphology of the mechanisms, by which the molecules experience the dissociation
SiC surface, while the annealing temperature determined the size and thermal hopping at the terrace as suggested by the scanning
of the resultant GQDs as the decrease in annealing temperature tunneling microscopy, while the C60 molecules fully decomposed
from 1,500 to 1,400◦ C increased the size of GQDs from 2.58 to at 650 K and transformed into uniform GQDs. They suggested
5.20 nm. that the annealing temperature determined the final shape of
the GQDs as thermal annealing at 725 K for 2 min resulted in
Stepwise Organic Synthesis/Cage Opening trapezoid-shaped, parallelogram-shaped, and triangular-shaped
Stepwise organic synthesis-mediated GQD fabrication is an GQDs (Figure 7C), whereas the hexagonal GQDs of around 5–
effective solution chemistry method, which offers uniform and 10 nm in diameter were obtained after thermal annealing at
well-defined GQDs. Despite the significant advancements in the 825 K for an additional 2 min. Similarly, Chen et al. oxidized
preparation of GQDs via stepwise organic synthesis, the poor C60 molecules following a modified Hummers method and
aqueous solubility of the produced GQDs and the possibility achieved ≈25 wt% yield of hexagonal graphene-oxide-like QDs
of large molecular size because of the side reactions are the (GOLQDs) (Figure 7D). The as-prepared GOLQDs were 0.6–
major bottlenecks, which need to be addressed (Ozhukil Valappil 2.2 nm in diameter, possessed an average thickness of 1.2 nm, and
et al., 2017). Further, the low throughput and the aggregation showed improved water solubility (Chen et al., 2015).
of GQDs in solution due to π-π interactions also demanded
careful considerations for industrial production. Mostly, the Chemical Vapor Deposition
interaction of aliphatic side chains with the aromatic molecules Chemical vapor deposition (CVD) is a well-known approach to
brings the graphene sheets closer to each other, thus triggering prepare 2D graphene. In a CVD technique, the flow rate of the
the GQD aggregation (Haque et al., 2018). Therefore, the hydrogen (H2 ) and carbon source, growth time, temperature, and
surface modification of the edges is an ideal way to avoid the surface morphology of the substrate, are the key parameters,
the aggregation by increasing the distance between graphene which determine the size of the ultimate product. By tuning
sheets, ultimately leading to improve the solubility of the GQDs. these parameters, the nucleation rate of the graphene could be
Following the solution chemistry method, Yan et al. used 3- speeded up to exceed the growth rate, leading to a decrease
iodo 4-bromo aniline as a precursor to fabricate three different- in the size of the final graphene product. The CVD-grown
sized GQDs containing different (170, 132, and 168) conjugated GQDs were first prepared by Fan et al. using copper foil as
carbon atoms (Figure 7A). They showed that the oxidative a substrate and methane as a carbon source, respectively (Fan
condensation of the precursor molecules during the stepwise et al., 2013). In their synthesis experiment, an oxidized surface
process lead to fused the graphene moieties, resulting in the layer on the copper foil was first removed by cleaning with
synthesis of GQDs, while, the modification of the edges by alcohol and HCl, followed by thermal treatment at 1,000◦ C in
′ ′
the covalent attachment of the 2 ,4,6 -trialkyl phenyl groups a furnace under a continuous supply of H2 and Ar at a flow
allowed the fabrication of water-soluble GQDs (Yan et al., rate of 10 ml/min and 200 ml/min, respectively. The H2 supply
2010). Notably, the covalent attachment of the phenyl groups was turned off after 40 min, while the H2 residues remaining in
at the edges significantly reduced the inter-layer distance of the reaction tube was removed by supplying Ar for a further
graphene as well as minimized the direct face to face contact 10 min. Afterward, the methane gas (CH4 ) was supplied for only
between graphene sheets, allowing improved aqueous solubility 3 s at a flow rate of 2 ml/min, followed by cooling of the copper
and the fabrication of stable GQDs. Subsequently, the same foil in an Ar environment. The resultant GQDs showed broad
group demonstrated the formation of heteroatom-doped GQDs size distribution (5–15 nm) as indicated by the DLS analysis,
(N-GQDs) by following the solution chemistry method (Li whereas the height profile (1–3 nm) suggested the formation
et al., 2012b). In their work, the N-GQDs were synthesized of few-layer-thick GQDs. Later, GQDs were prepared without
by a two-step process. First, a nitrogen-containing intermediate any metal catalyst by Ding et al. using hexagonal boron nitride
was prepared using small substituted benzene derivatives as a as a substrate (Ding, 2014). By tuning the ratio of different
precursor such as phenylboronic acid and 2-bromo-5-iodo-30- gases, e.g., Ar:CH4 :H2 , the GQDs were fabricated under constant
(phenylethynyl)-1,10-biphenyl, followed by the Suzuki coupling reaction time, which possessed a different number of graphene
of the intermediate molecule to form a GQD precursor. In the and exhibited thickness-dependent PL in the visible region.
second step, the GQDs precursor was treated with an excess Following the CVD approach, the direct formation of single
amount of iron (III) chloride under an inert environment (argon, crystalline GQDs on silicon wafer was also demonstrated (Huang
Ar) in a mixture of nitromethane/dichloromethane to yield N- et al., 2016). The as-fabricated GQDs were round shaped, 5–
GQDs (Figure 7B). The as-obtained N-doped GQDs showed 10 nm in size, and 2 nm thick, while they possessed an average
excellent electrocatalytic ORR activity. thickness of 1.2 nm, which suggested single- to few-layer-thick
Interestingly, the possibility of graphene wrapping into quasi GQDs. Recently, Nessim’s group used chitosan, a non-toxic and
0-D fullerene GQDs provided a new concept to develop well- cheap biopolymer, as a source of C and N to fabricate N-GQDs
ordered GQDs from the fullerene via cage opening. For instance, (Kumar et al., 2018). The DLS analysis (10–15 nm) and the AFM
Lu et al. used fullerene as a precursor and ruthenium (Ru) as height profile (2–5 nm) suggested the formation of multilayer-
a catalyst to rupture the C60 cage, leading to the formation thick N-GQDs. They proposed that during the synthesis process,
of GQDs (Lu et al., 2011). As a reaction process, the thermal the chitosan first decomposed into N-containing compounds,

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FIGURE 7 | (A) GQDs containing different (170, 132, and 168) conjugated carbon atoms; reproduced from Yan et al. (2010) with permission from the American
Chemical Society. (B) The representation of three different colloidal GQDs prepared by solution chemistry; reproduced from Li et al. (2012b) with permission from the
American Chemical Society. (C) STM images represented the different shapes of GQDs prepared by cage opening approach; reproduced from Lu et al. (2011) with
permission from Nature Publishing Group. (D) Illustration of the process of graphene-oxide-like QDs (GOLQDs) formation by the oxidization of C60 ; reproduced from
Chen et al. (2015) with permission from Wiley.

followed by the adsorption and nucleation of the HCN species probe for monitoring the cellular dynamics as well as in vitro
to form photoluminescent N-GQDs (Figure 8). Interestingly, and in vivo tumor imaging. A redox-sensitive fluorescent probe
the PL emission in the visible region highlighted the potential based on GQDs was devised by Li et al. (2016a). By using this
applications of N-doped GQDs in nano-optoelectronic. probe, the reductive or oxidative stress-induced dynamic changes
in the intracellular redox state were monitored in real time.
GQDs FOR BIOIMAGING APPLICATIONS Notably, the potential of GQDs for determining the cellular
dynamics was demonstrated in this study for the first time.
Previously, organic dyes and inorganic semiconductor QD-based Besides the conjugation of GQDs with the recognition elements
fluorophores have been usually applied for cellular visualization or proteins, Chen’s group later reported the functionalization of
and bioimaging, respectively. However, the photobleaching and GQDs with monosaccharide sugar to determine the trafficking
the low extinction coefficient of organic dyes, and the poor and the overall distribution of cell surface carbohydrate receptor
water solubility and the intrinsic toxicity of the semiconductor (Chen et al., 2017b). Recently, the same group further performed
QDs, are the main obstacles toward their practical bioimaging the real-time estimation of change in the level of intracellular
applications. Being a 0D member of the carbon family, GQDs hydrogen sulfide (H2 S) in live cells (Li et al., 2018b). Specifically,
hold great promise to actively substitute these fluorophores the surface functionalization of the GQDs with dinitrophenyl
owing to the tunable and strong PL, photostability, excellent (an electron-withdrawing group) significantly quenched the PL
biocompatibility, and effective renal clearance, thus offering of GQDs due to the light-induced electron transfer, whereas
unprecedented opportunities for bioimaging (Zheng et al., the cleavage of these groups by H2 S recovered the GQD PL.
2015). In the following sub-sections, the potential bioimaging Hence, a positive correlation has been noticed between the
applications of GQDs, including fluorescence imaging, two- PL intensity and the intracellular H2 S level. Compared to
photon imaging, magnetic resonance imaging, and dual-modal the previously developed GQD-based fluorescent probes, the
imaging, will be discussed. designed fluorescence turn-on strategy ensures the selective
determination of H2 S.
Fluorescence Imaging Instead of monitoring the cellular dynamics, Gao et al.
Since the first demonstration of fluorescent GQDs by Pan et al. presented polyethyleneimine (PEI)-coated GQDs for in vitro
(2010), GQDs have been actively developed as a fluorescent tumor cell imaging (Gao et al., 2017). Depending upon the

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FIGURE 8 | Schematic representation of the N-GQD fabrication via chemical vapor deposition (CVD) process; reproduced from Kumar et al. (2018) with permission
from the American Chemical Society.

molecular weight (MW) of the PEI, the prepared GQDs exhibited negatively charged cellular membrane greatly facilitated the
red, yellow, and blue emission, allowing multicolor imaging of efficient internalization, resulting in a five-fold higher cellular
U87 tumor cells in vitro. They suggested that the coating of PEI uptake of PNF-GQDs than GQDs alone. Subsequently, folic
not only determined the core structure of GQDs but also altered acid-conjugated GQDs (FA-GQDs) were also reported by Zhang
the energy gap, resulting in multicolor emissive GQDs. Instead et al. for targeted FL imaging (Zhang et al., 2019a). The time-
of larger and few-layer-thick GQDs, monolayer ultrasmall dependent enhanced fluorescence of FA-GQDs was observed
GQDs were prepared following the pyrolysis approach, showing in SKOV3 cells (Figure 9C), which confirmed the selective
excitation wavelength-independent blue PL with a QY of 3.6%. internalization due to FA targeting, whereas the confocal
Notably, due to the ultrasmall size, GQDs effectively penetrated fluorescence microscopy indicated the positive correlation
into the nuclei of HeLa cells as monitored by tracking the blue between the internalization of FA-GQDs and the expression of
fluorescence of GQDs during FL imaging (Hong et al., 2018). cell surface FA receptor. Though an enhanced targeting imaging
Considering the water solubility, excellent biocompatibility, and has been achieved, Bansal et al. reported that the conjugation
non-toxicity of GQDs, Ding et al. developed a GQD-based of targeting molecules decreased the PL yield of GQDs (Bansal
theranostic nanoagent loaded with doxorubicin (DOX) (Ding et al., 2019). They developed plain GQDs conjugated with either
et al., 2017). The internalization of the nanoagent was tracked biosurfactant or FA receptor. The PL of plain GQDs significantly
by the blue fluorescence emitted from the GQDs, while due reduced from 12.8 to 10.4% for biosurfactant-GQDs and 9.08%
to the close proximity, the DOX fluorescence was significantly for FA-GQDs. They suggested that the chemical interactions
quenched by GQDs. However, a bright green fluorescence and local electric field occurred on the GQD surface during
is observed from DOX after internalization, which indicated bioconjugation leading to a change in the electronic energy
the effective release of DOX from the nanoagent, resulting of GQDs, resulting in a decreased photoluminescence QY of
in substantial chemotherapeutic killing and significant tumor conjugated GQDs.
inhibition (Figure 9A). In contrast to GQDs, heteroatom-doped GQDs have also
Meanwhile, the release of red fluorescent Cy 5.5 dye from been employed for cellular imaging. For instance, Wang’s group
the nanoagent in response to the overexpression of cathepsin D reported the efficient labeling of HepG2 cells by green fluorescent
molecule further confirmed the higher chemotherapeutic killing. N-doped GQDs (Li et al., 2018a). In addition, N-GQDs also
Besides the non-selective uptake and cellular imaging, protein served as a fluorescent probe for optical sensing of formaldehyde
nanofiber-conjugated GQDs (PNF-GQDs) offered targeted due to the redox-sensitive fluorescence turn on/off. Similarly, B-
fluorescence imaging due to the attached RGD receptor as a doped GQDs with excitation-independent PL were prepared by
targeting moiety (Su et al., 2015). Owing to the effective targeting, Hai et al. following the one pot acid-free approach (Hai et al.,
HeLa cells exhibited far bright fluorescence signal of PNF- 2015). A bright blue PL under 360-nm excitation was observed in
GQDs than CO-7 cells as evidenced by confocal microscopy, Hela cells incubated with B-GQDs, which suggested the potential
suggesting the preferential cellular uptake capacity of targeted of as-obtained B-GQDs for cellular imaging. Later, Wang
PNF-GQD probe (Figure 9B). On the other hand, the strong et al. presented orange fluorescent phosphorus-doped GQDs (P-
electrostatic interaction between positively charged PNF and GQDs) and blue fluorescent B-GQDs, for in vitro imaging (Wang

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FIGURE 9 | (A) Strategy of in vivo monitoring of drug and tumor therapy by GQD-based theranostic agent; reproduced from Ding et al. (2017) with permission from
the American Chemical Society. (B) Confocal fluorescence (FL) images of HeLa cells incubated with GQDs (i), PNF-GQDs (ii), and CO-7 cells incubated with protein
nanofiber-conjugated GQDs (PNF-GQDs) (PNF-GQDs) (iii); reproduced from Su et al. (2015) with permission from Wiley. (C) Confocal FL images of BMSC (i), MCF7
(ii), MDA-MB-231 (iii), and SKOV3 (iv) under ultraviolet (UV) light irradiation, reproduced from Zhang et al. (2019a) with permission from Wiley.

et al., 2019a). Owing to the matched energy level, they suggested et al., 2018). In contrast, Campbell et al. reported multicolor
the rapid energy transfer from B-GQDs to P-GQDs, which led emissive S-doped, N-doped, and B,N-co-doped GQDs for both
to an increase in the QY of P-GQDs. Recently, visible light- visible and NIR-I imaging in vitro (Campbell et al., 2019). Under
emitted S-GQDs have also been used for cellular imaging (Jin different excitation wavelengths, these doped GQDs emitted blue

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(450 nm), green (535 nm), and red (750 nm) light (Figure 10A). photodynamic antibacterial therapy (TP-PDT). Similarly, amino-
Notably, this multicolor emission is attributed to the quantum functionalized N-GQDs (amino-N-GQDs), as a dual-modal
size of GQDs and the electronic state or the arrangements of agent for TPFI and TP-activated antibacterial PDT of multi-
the surface defects. Moreover, the pH-dependent fluorescence drug resistant bacteria, were developed by Kuo et al. (2018).
emission of these GQDs also allowed the ratiometric detection Importantly, amino functionalization and the N-doping changed
of healthy (HEK-293 cell) and tumor cells (HeLa and MCF-7 the intrinsic electronic and optical properties of GQDs, resulting
cell). Similarly, heteroatom co-doped GQDs such as P,N and Fe,N in high TPA cross-section (54,356 GM) and a QY of 0.33, of
co-doped GQDs were also demonstrated for cellular imaging by the as-obtained amino-N-GQDs. In a subsequent study, amino
different research groups (Ananthanarayanan et al., 2015; Gao and sulfo co-functionalized GQDs were also demonstrated for
et al., 2018). Besides in vitro cellular imaging, Wang’s group TPFI (Wang et al., 2018b). Though these GQDs showed little low
developed red fluorescent GQDs and demonstrated NIR-I in vivo TPA cross-section (38,000 GM), the ultra-high stability due to the
imaging (Ge et al., 2014). After sub-cutaneous injection, GQDs edge functionalization suggested their suitability for long-term
exhibited much higher FL intensity at the injection site than TP cellular imaging. Recently, Singh et al. reported on in vitro
mouse skin without any apparent decay (Figure 10B). However, and in vivo TPFI using hydrothermally synthesized GQDs from
the fabrication process of GQDs is quite lengthy, laborious, and neem extract (Singh et al., 2019). A bright green luminescence
complicated. Compared to NIR-I, in vivo imaging in the NIR-II of GQDs from RAW cells under TPE at 900 nm indicated
region was recently established by Zhang and coworkers using the internalization of GQDs in lysosome, as further evidenced
N,B-co-doped GQDs, which exhibited broad PL emission (950– by LysoTracker (Figure 11A). In addition, in vivo two-photon
1,100 nm) (Wang et al., 2019b). Under 808-nm laser excitation, microscopy (TPM) of zebrafish embryo and larvae showed strong
a bright fluorescence signal of intravenously injected N,B-co- fluorescence of GQDs in a yolk sac region compared with other
doped GQDs was clearly observed in the liver and kidneys of tissues, confirming the potential of GQDs as a contrast agent for
the mice. Moreover, the designed GQDs also allowed an efficient TPFI of the digestive system (Figure 11B). Peptide-conjugated
visualization of the blood vessels (Figure 10C). Collectively, N-B- GQDs also showed enhanced penetration into MCF-7 cells as
GQDs showed great potential as an NIR-II nanoprobe for organ monitored by the TPFI, but they showed 6,500 GM TPA cross-
and vasculature imaging in vivo. section, which is much lower than other GQD-based TPFI
contrast agents (Sapkota et al., 2017). In addition to TP cellular
imaging, GQDs have also been employed by different researchers
Two-Photon Imaging for the in vitro and in vivo TPFI of biomolecules. For example,
Two-photon fluorescence imaging (TPFI) has attained enormous Zhao et al. demonstrated the in vitro and in vivo TPFI of H2 O2
attention because of larger tissue penetration, low signal- (Zhao et al., 2016b), while Tan’s group used GQDs for the in vitro
to-noise ratio, minimum background autofluorescence, less TPFI of ascorbic acid (Feng et al., 2017).
photobleaching, and reduced photoinduced toxicity, thus Like GQDs, heteroatom-doped GQDs also showed potential
holding great promise in biomedical research and diagnostics as a contrast probe for TPFI. For example, Liu et al. prepared N-
than single-photon Fl imaging (Yoo et al., 2015). Compared to GQDs via solvothermal approach for deep TPFI of cells/tissues
continuous wave (CW) excited one-photon Fl imaging (OPFI), (Liu et al., 2013b). In this work, they used DMF as both an N
TPFI offered several advantages: (1) the detailed monitoring of source and a solvent, and the resultant N-GQDs showed high
deeply occurring biological activities within the body; (2) high TPA cross-section (48,000 GM). Under femtosecond pulsed laser
spatiotemporal resolution and reduced photobleaching due to (800 nm) excitation, HeLa cells stained with GQDs exhibited
the femtosecond pulsed laser excited two photons via non-linear bright blue fluorescence. Interestingly, compared to the OPFI
excitation; (3) Fl imaging of deeply residing organelles/tissues as (400 µm), the TP fluorescence signal was detected even at a
well as the diagnosis of deep-seated tumors owing to the two- tissue thickness of 1,800 µm, which suggested the potential of
photon excitation in both first and second biological window N-GQDs for deep cellular imaging at least within 800–1,500 µm
(700–1,350 nm) (Lin et al., 2016). thickness. Later, single-layer-thick (0.83 nm) polymer-conjugated
Recently, GQDs have emerged as a promising candidate for N-GQDs were developed by Wu et al. for TPFI (Wu et al., 2018).
TPFI and surpassed the conventional two-photon fluorophores As N-doping and the surface passivation of GQDs by polymer
because of high two-photon absorption (TPA) cross-section and conjugation significantly trapped the emissive energy on the
excellent photostability. For instance, Pu’s group reported the surface, the N-GQDs exhibited high TPA cross-section (57,980.1
ultrasonic preparation of TP fluorescent GQDs with a TPA GM) and impressive QY (0.573), along with high PL emission
cross-section of 47,903 Goppert–Mayer units (GM, and 1 GM intensity in an acidic condition. Owing to high TPA cross-section
= 10−50 cm4 s photon−1 ) in the NIR-I region and a QY of and TP excitation (TPE) at 800 nm, N-GQDs allowed TPFI in the
0.187 (Kuo et al., 2016). Under TP excitation (TPE, 800 nm) at near-infrared I (NIR-I) region as 35-fold brighter TP signal was
a power of 0.74 mW, the TP luminescence emitted from GQDs observed in N-GQD-labeled A431 skin cells than unlabeled cells
revealed the clear localization of GQD-treated gram-positive and under a low TPE power (0.598 mW) (Figure 11C).
gram-negative bacteria, even at a depth of 75 µm. Along with
TPFI, the designed GQDs also induced substantial photodynamic Magnetic Resonance Imaging (MRI)
antibacterial killing under pulsed laser excitation (2.64 mW) for Being a highly sensitive and non-invasive technique, MRI
only 15 s, proving their potential as a photosensitizer for TP has emerged as a state-of-the-art imaging modality, offering

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FIGURE 10 | (A) Representation of heteroatom-doped GQDs for multicolor imaging and cancer cell detection; reproduced from Campbell et al. (2019) with
permission from the American Chemical Society. (B) Bright-field (i) and (ii) in vivo red-fluorescence image of GQDs after subcutaneous injection; reproduced from Ge
et al. (2014) with permission from Nature Publishing Group. (C) NIR-II in vivo imaging of live mice in supine position; reproduced from Wang et al. (2019b) with
permission from Elsevier. (a) Digital image of a nude mouse. (b,c) NIR-II imaging of a control either without injection or with only PBS. (d–i) NIR-II imaging of nude mice
at mentioned time points after an i.v. injection of N,B co-doped GQDs (1 mg/ml, 200 L). (j) Higher magnified NIR-II images captured at indicated time points, showing
blood vessels in the head. The PBS group was used as a control.

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FIGURE 11 | (A) In vitro co-localization study using Lyso Tracker to monitor the internalization of the GQDs in RAW cells. (B) Bright-field (left) and FL images (right) of
zebra fish embryos and larvae treated with GQD solution for 30 min, and then imaging at different time-points (i) 1 hpf, (ii,iii) 72 hpf, and (iv) 96 hpf. (ii) the control;
reproduced from Singh et al. (2019) with permission from the Royal Society of Chemistry. (C) Depth and laser power-dependent TPFL images of N-GQDs labeled (i)
and unlabeled A431 cells (ii); reproduced from Wu et al., 2018) with permission from the Royal Society of Chemistry.

high spatiotemporal resolution and deep tissue penetration glycol (PEG) functionalized Gd-1,4,7,10-tetraazacyclododecane-
(Vijayalaxmi et al., 2015). Impressively, these features allowed 1,4,7,10-tetraacetic acid (Gd-DOTA) complex (Yang et al., 2019).
quantitative interrogation of diverse cellular events, tissues Different PEG chains were used for functionalization, and the
dynamics, and cellular anatomy. On the other hand, compared highest longitudinal relaxivity was achieved from GQD-PEG12 -
to ionizing radiation-based imaging modalities such as positron Gd, which was about 16-fold higher than the commercial MRI
emission tomography (PET) and computed tomography (CT), contrast agent (Gd-DTPA). They suggested that the PEG chains
MRI is a non-ionizing radiation-based approach, thereby strongly influenced the Gd3+ rotation, and thus, the longitudinal
avoiding the radiation-induced damages and toxicity. Besides the relaxivity could be effectively regulated by controlling the
potential advantages, the long operational time of MRI for signal chain length (Figure 12A). After intravenous (i.v.) injection
acquisition as well as its low sensitivity (∼10−3 -10−5 mol/L) into A549 tumor-bearing mice, the hyaluronic acid-targeted
required careful considerations for practical clinical applications PGQDs (PGQDs-HA) selectively entered into the tumor and
(Revia and Zhang, 2016). showed significantly enhanced MRI signal after 2 h post-injection
In MRI, the T1 relaxation time (longitudinal) and the T2 (Figure 12B). In addition, owing to the DOX loading, PGQDs
relaxation time (transverse) of the magnetic moment of water induced substantial chemotherapeutic killing, which suggested
proton are usually determined. It has been observed that the that MRI-guided cancer therapy could be achieved using PGQD-
magnetic moment of water proton is environment dependent, based theranostic platform. Though an improved MRI efficiency
and thus, different attempts have been made to enhance the has been demonstrated, the toxicity associated with Gd raises
relaxivity, which directly led to improve either T1 or T2 serious safety concerns.
contrast. Generally, gadolinium (Gd3+ ) and iron oxide were Alternatively, Zhang’s group reported paramagnetic metal-
employed as a T1 and T2 contrast agents, providing bright and free B-GQDs as an MRI contrast agent with a longitudinal
dark MRI images, respectively. However, these contrast agents relaxivity (r1 ) of 18.277 mm−1 s−1 (Wang et al., 2017). The
are more or less toxic due to the non-specific conjugation paramagnetic characteristic was attributed to the introduction
with biological molecules. In addition, the T1 contrast agent of vacancies and elemental boron via doping. Meanwhile, the
(Gd3+ ) exhibited little selectivity, low relaxivity, and short much higher r1 compared with the un-doped GQDs (0.0038
circulation time, which significantly decreased the efficiency of mm−1 s−1 ) and even commercial Gd-DTPA (5.39 mm−1
MRI. Mostly, Gd3+ was incorporated into different carriers s−1 ) confirmed the doping enhanced longitudinal relaxivity.
to enhance the relaxivity and delayed the circulation time Moreover, the B-GQDs also showed substantial time-dependent
(Wang and Zhou, 2016). Among different carriers, GQD is contrast enhancement in vivo after subcutaneous injection
an attractive nanocarrier owing to its biocompatibility, water (Figure 12C). Thus, B-GQDs are a standalone and safer
solubility, and non-toxicity. Recently, Yang et al. developed alternative to previously developed Gd-based contrast agents,
paramagnetic GQDs (PGQDs) by incorporating polyethylene holding great potential for clinical MRI.

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Younis et al. Recent Advances on Graphene Quantum Dots

FIGURE 12 | (A) Influence of polyethylene glycol (PEG) chains on the longitudinal relaxivity. (B) In Vivo T1 -weighted MR images of A549 tumor-bearing mice before
and after injection of hyaluronic acid-targeted PGQD (PGQD-HA); reproduced from Yang et al. (2019) with permission from the American Chemical Society. (C) In Vivo
T1 -weighted MR images of mice before and after i.v. injection of B-GQDs; reproduced from Wang et al. (2017) with permission from Wiley. The arrows represented
different organs: heart (H), kidney (K), stomach (St), and spleen (Sp).

Dual-Modal Imaging contrast agents have been developed for dual-modal Fl/MRI,
The concept of integrating multiple imaging modalities Fl/PAI, and Fl/OCT imaging. For example, Huang et al.
into one system has gained much popularity, recently (Fan developed paramagnetic GQD-based theranostic platform for
et al., 2017). By combining the advantages of individual dual-modal (Fl/MRI) imaging and targeted chemotherapy
imaging modality, dual-modal imaging offers highly efficient (Huang et al., 2015). In their work, the paramagnetic
and accurate tumor diagnosis (Huang et al., 2014). On the GQDs were first fabricated by the covalent conjugation of
other hand, compared to the administration of different diethylenetriaminepentaacetic acid gadolinium (Gd-GQDs) and
contrast agents for individual imaging, the introduction then decorated with FA receptor (folate-GdGQDs) to achieve
of multiple imaging functionalities into a single agent targeted imaging guided chemotherapy by the loaded DOX.
greatly avoids the stress on the body as well as promotes Under visible light, confocal microscopic (CLSM) analysis
an effective clearance of such dual-modal contrast agent revealed the bright green fluorescence of folate-GdGQDs
(Cheng et al., 2012). internalized into HeLa cells, confirming their selective uptake
Owing to the intrinsic fluorescence, GQDs have been via receptor-mediated endocytosis as well as in vitro imaging
actively integrated with other imaging modalities such as capacity (Figure 13A). Meanwhile, the higher longitudinal
MRI, photoacoustic imaging (PAI), and optical coherence relaxivity (r1, 11.49 mm−1 s−1 ) and transverse relaxivity
tomography (OCT), and thus, the different GQD-based (r2, 28.79 mm−1 s−1 ) owing to Gd conjugation allowed the

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Younis et al. Recent Advances on Graphene Quantum Dots

FIGURE 13 | (A) In Vitro internalization of folate-GdGQDs in HeLa cells. (B) T1 and T2 -weighted MRI images of diethylenetriaminepentaacetic acid gadolinium and
then decorated with FA receptor (folate-GdGQDs) at various Gd concentrations (i) and T1 -weighted MRI images of HeLa cells incubated with various concentration of
folate-GdGQDs (ii); reproduced from Huang et al. (2015) with permission from the Royal Society of Chemistry.

enhanced T1 and T2 -weighted MRI (Figure 13B). Along with Subsequently, instead of G-doped GQDs, superparamagnetic
dual-modal imaging, folate-GdGQDs selectively delivered GQDs (MGQDs) were developed by Justin et al. for dual-
the DOX, leading to significant chemotherapeutic killing modal Fl/MRI (Justin et al., 2016). Iron oxide nanoparticles
in vitro. Hence, folate-GdGQDs showed great promise as a (Fe3 O4 ) were incorporated onto GQDs via a facile approach,
nanotheranostic agent for dual-modal imaging-guided targeted and the resultant MGQDs exhibited PL emission at 398 nm
cancer chemotherapy. and a T2 relaxation time of 4.16 mm−1 s−1 with a strong

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Younis et al. Recent Advances on Graphene Quantum Dots

FIGURE 14 | (A) Visible light, confocal microscopic (CLSM) images of superparamagnetic GQDs (MGQDs) labeled (i) and unlabeled (ii) 3T3 cells. (B) magnetomotive
optical coherence tomography (OCT) images of MGQDs labeled (i) and unlabeled (ii) 3T3 cells; reproduced from Li et al. (2019) with permission from Wiley. (C,D)
Concentration-dependent enhanced near-infrared (NIR) fluorescence as well as photoacoustic imaging (PAI) signal. (E) In Vitro PAI intensity of FA-N-GQDs and
N-GQDs in both HeLa cells and A549 cells, respectively; reproduced from Xuan et al. (2018) with permission from IOP Publishing. **means P value is 0.05.

dependence on Fe concentration. Meanwhile, a concentration- for OCT, the MGQD-labeled cells were placed on an agar
dependent enhanced PL signal of MGQDs was observed from gel to mimic the human soft tissues, and then imaged
glioblastoma cells, which suggested an efficient in vitro imaging. by an in-house magnetomotive OCT system. Compared to
Besides Fl/MRI contrast agent, MGQDs also served as a drug unlabeled 3T3 cells, a significant magnetomotive signal (320 Hz)
nanocarrier as well as a photothermal agent, presenting dual- was observed from MGQD-labeled cells, which allowed the
modal imaging-guided synergistic chemo/photothermal tumor successful cellular visualization by OCT (Figure 14B). Hence,
therapy. In another study, targeted dual-modal Fl/MRI-guided the intrinsic fluorescence and superparamagnetic property
chemotherapy by Fe3 O4 @SiO2 @GQD-FA was also demonstrated make MGQDs a promising contrast agent for dual-modal
(Su et al., 2017). The PL emission at 460 nm and an enhanced Fl/OCT imaging. Recently, using 131 I-labeled FA-GQDs, Wei’s
transverse relaxation time (r2 , 62.8 mm−1 s−1 ) for T2 -weighted group demonstrated targeted single-photon emission computed
MRI enabled an efficient guidance of chemotherapy by dual- tomography (SPECT) imaging in vivo (Wang et al., 2019c).
modal Fl/MRI in vitro. Owing to FA targeting, the 131 I-labeled FA-GQDs were
In contrast to Fl/MRI, Li et al. reported on superparamagnetic preferentially uptaken by the HeLA cells, while after i.v.
GQDs (MGQDs) as a dual-modal contrast agent for Fl/optical into mice, the in vivo SPECT imaging revealed their higher
coherence tomography imaging (Fl/OCT) (Li et al., 2019). accumulation in the liver and tumor region, compared to those
The MGQDs internalized into 3T3 exhibited blue fluorescence of non-conjugated GQDs. Importantly, the biodistribution study
as revealed by in vitro CLSM imaging (Figure 14A), while confirmed their effective distribution and rapid metabolization

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TABLE 1 | Toxicity of graphene quantum dots (GQDs) and doped-GQDs.

Material Cells Assay Toxicity Incubation time References

GQDs MCF-7, Hela, MCF-10A MTT >95% 24 h Roy et al., 2015

(2 mg/ml)
GQDs Hela, A549 MTT LDH >95% 24 h Chong et al., 2014
(160 µg/ml) >85%
(640 µg/ml)
GQDs Hela CKK-8 90% 24 h Jiang et al., 2015
(100 µg/ml)
GQDs A549 MTT >80% 24 h Yuan et al., 2014
(200 µg/ml)
GQDs KB, MDA-MB231, A549 MTT >95% 21 days/24 h Nurunnabi et al., 2013
(500 µg/ml) MDCK LDH
GQDs A549 MTT 80% 24 h Sun et al., 2013
(100 µg/ml)
GQDs MGC-803 MCF-7 MTT GQDs < GO 3 days Wu et al., 2013
GQDs Stem cells MTT >70% 24 h Zhang et al., 2012
(200 µg/ml)
GQDs Stem cells MTT 61% 24 h Qiu et al., 2016
(100 µg/ml)
GQDs THP-1 macrophages MTT 82.5% 24 h Qin et al., 2015b
(200 µg/ml)
GQDs MG-63 MC3T3 MTT >80% 24 h Zhu et al., 2011
(400 µg/ml)
GQDs RSC96 MTT 70% 24 h Zhu et al., 2015c
(200 µg/ml)
B-GQDs Hela MTT 87% 24 h Hai et al., 2015
(4 mg/ml)
N-GQDs Red blood cells (RBC) Hemolysis ATP N-GQDs < GO 12 h Wang et al., 2015

after 48-h post-injection, suggesting the rapid clearance from et al., 2011), the detailed toxicity investigations prior to their
the body to ensure the non-toxicity of the GQDs. Owing introduction into the biological systems are mandatory to ensure
to efficient SPECT imaging, the designed GQDs would be a their non-toxic biological effects. It has been noticed that
suitable candidate for the SPECT imaging of FA-overexpressed the surface charge, functional groups, and the size of GQDs
tumor. Dual-modal Fl/PAI imaging using N-GQDs was reported determine the toxicity of the QDs (Schroeder et al., 2016).
by Xuan et al. for the first time (Xuan et al., 2018). The Compared to pristine GQDs, PEGylated GQDs showed less
developed N-GQDs exhibited fluorescence emission in both toxicity due to the biocompatible PEG molecule. Notably, the
visible and NIR regions. Meanwhile, a concentration-dependent PEG surface functionalization led to an increase in the size
enhanced NIR fluorescence as well as PAI signal were observed of the resultant GQDs as well as it imparts a hydrophilic
(Figures 14C,D), which suggested the linear relationship (R2 = character, resulting in reduced toxic effects and safer biological
0.9969). After FA functionalization, FA-N-GQDs were preferably imaging applications (Chandra et al., 2014). We have presented
uptaken by the HeLa cells than A549 cells as revealed by in the in vitro and in vivo toxicity of GQDs in Table 1.
vitro PAI (Figure 14E) and triggered significant photothermal For thorough understanding, the readers are referred to the
destruction of tumor cells due to the PTT effect. These findings comprehensive and state-of-the-art recently published reviews
strongly conferred that the targeted tumor PTT could be (Schroeder et al., 2016; Wang et al., 2016a; Selestin Raja et al.,
achieved under the guidance of dual-modal Fl/PAI by FA- 2019), which provided the elaborative and in-detail review of
conjugated N-GQDs. GQD toxicity.
Chong et al. determined the toxicity of PEG-GQDs onto HeLa
TOXICITY OF GQDs cells and A549 cells by LDH assay and WST-1, respectively
(Chong et al., 2014). They suggested no apparent cytotoxicity
The inherent toxicity of inorganic nanomaterials presents as more than 80% of the cell viability was recorded for both
prominent obstacles to their potential biomedical applications. HeLa and A549 cells even at a high concentration (160 µg/ml
Therefore, the in vitro and in vivo toxicity of GQDs should for HeLa and 640 µg/ml for A549). Moreover, compared to
be clearly understood to advance their practical bioimaging the control, a comparable ROS level and LDH were observed,
applications. Though GQDs belong to a family of carbon which confirmed that PEG-GQDs neither disrupt the cell
nanomaterial, which predicts their low cytotoxicity (Kakran membrane integrity nor induce oxidative stress. In contrast

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Younis et al. Recent Advances on Graphene Quantum Dots

to cellular toxicity, Wu et al. conducted the genotoxicity confinement effect, surface modification, edge state, doping,
investigations of GQDs by performing cell cycle analysis (Wu size effect, etc., a clear and detailed mechanism of PL with
et al., 2013). They found that GQDs slightly damaged the sufficient interpretation is still missing. Hence, the integration
cellular DNA of MCF-7 and MGC 803 cells, but this was of experimental investigations with theoretical measurements is
still far superior than GO, which triggered significant cell greatly needed to provide a deeper and thorough understanding
accumulation in the sub-G1 phase, resulting in increased of the PL mechanism. (3) Most of the reported GQDs exhibited
cell apoptosis. narrow spectral PL emission either in the blue or green
Apart from in vitro testing, Nurunnabi et al. demonstrated fluorescence, thus seriously suffering from low penetration depth
that i.v.-injected carboxylated GQDs exert very low toxicity and and high tissue scattering, respectively. However, the formation
organ damage even after 21 days as verified by complete blood of GQDs having an NIR I/II emission is a formidable challenge as
counts, histological analysis, and blood biochemistry (Nurunnabi the QY of GQDs in the NIR region is considerably low, which
et al., 2013). In another report, Chong et al. suggested that limited the further advancement of GQDs as a contrast agent
intravenous and intraperitoneal injection of multiple doses of for bioimaging. Owing to the minimum auto-fluorescence and
PEG-GQDs (20 mg/kg every second day) into mice for 14 low light absorption by the tissues in the NIR region, GQDs
days did not induce any noticeable toxicity compared to the with NIR-I or NIR-II emission will be a potential candidate for
control (Chong et al., 2014). Similarly, no apparent toxicity of in vivo imaging of deep-seated tumors, whereas with the rapid
GQDs and gadolinium-doped FA functionalized GQDs (Gd- development of new and innovative fabrication strategies, the
FA-GQDs) were observed in zebrafish (Huang et al., 2015). NIR I/II-emitted GQDs with high QY could be expected in the
However, Gd-FA-GQDs induced little toxicity at a higher near future for precise and accurate tumor diagnosis.
concentration (500 µg/ml), which is attributed to the release
of Gd ions from the GQDs. This implies that the toxicity CONCLUSION
of the doping element as well as the stability of the overall
GQD complex should be taken into consideration to ensure In this review, we have attempted to provide a comprehensive
biological safety. account of the latest cutting-edge advancements in GQD research
with a special emphasis on their bioimaging applications.
CURRENT CHALLENGES AND FUTURE The recent progress in fabrication strategies including top–
PROSPECTS down and bottom–up has been critically reviewed, following
an in-detail discussion on the potential in vitro and in vivo
Despite the significant advancements and the proven remarkable bioimaging applications of GQDs. A comparative and balanced
advantages, the potential bioimaging applications of GQDs viewpoint has been given, which may assist to overcome the
have not been fully explored yet owing to certain unresolved existing odds, and facilitates the design of next-generation GQD-
challenges. (1) Although GQDs have been fabricated by a number based contrast agents for clinical diagnostics. We believe that
of methods as discussed in the Synthesis Strategies of GQDs this timely rigorous account offers an in-depth understanding,
section, the controlled formation of high-quality single-layer which will promote more exciting and innovative developments
GQDs with narrow size distribution is still a challenging task. in the future, leading to shift in the GQDs from bench
Meanwhile, most of these reported methods usually suffer from to bedside.
long reaction time, toxic organic solvents, etc., and provide
considerably low product yield. As the optical and electronic AUTHOR CONTRIBUTIONS
properties of GQDs strongly depend on their size and shape, the
controlled formation of GQDs is an ideal way to enhance their MY wrote the manuscript. GH helped in designing the schematic.
intrinsic physicochemical properties; therefore, the exploration MY, JL, and PH discussed and revised the manuscript. All authors
of a controllable, high-yield, and environment friendly method contributed to manuscript revision, read, and approved the
for GQD fabrication is highly desirable yet quite advantageous submitted version.
for the large-scale industrial production of high-quality GQDs
for biomedical applications. (2) The designed GQDs exhibited FUNDING
very low QY for green and red fluorescence, which is even lower
than the organic fluorophores and conventional semiconductor This work was financially supported by the National Key
QDs. This low QY especially in the NIR region presented a Research and Development Program (2018YFA0704003), the
potential barrier toward the practical applications of GQDs for National Natural Science Foundation of China (31771036,
tumor imaging. Meanwhile, the alteration of optical properties 51703132), the Basic Research Program of Shenzhen
by surface passivation or heteroatom doping could enhance (JCYJ20180507182413022, JCYJ20170412111100742), the
the fluorescence QY of GQDs. On the other hand, the poor Guangdong Province Natural Science Foundation of Major Basic
understanding regarding the PL mechanism of GQDs seriously Research and Cultivation Project (2018B030308003), and the
limited the further improvement in QY. Despite the several Fok Ying-Tong Education Foundation for Young Teachers in
proposed mechanisms of PL, including surface state, quantum the Higher Education Institutions of China (161032).

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Frontiers in Chemistry | www.frontiersin.org 25 June 2020 | Volume 8 | Article 424