Vol.

Frontiers in Nanobiomedical Research

THE WORLD SCIENTIFIC ENCYCLOPEDIA OF

NANOMEDICINE AND BIOENGINEERING II
Bioimplants, Regenerative Medicine, and
Nano-Cancer Diagnosis and Phototherapy

 Synthesis and Biomedical Applications

of Graphene Quantum Dots

9559_9789814667647_TP_V1.indd 1 14/7/17 9:13 AM

 Frontiers in Nanobiomedical Research
ISSN: 2251-3965

Series Editors: Martin L. Yarmush (Harvard Medical School, USA)

Donglu Shi (University of Cincinnati, USA)

Published
Vol. 6 Handbook of Immunological Properties of Engineered Nanomaterials
(In 3 Volumes)
edited by Marina A. Dobrovolskaia and Scott E. McNeil
(Leidos Biomedical Research Inc., USA)

Vol. 7 Multiscale Technologies for Cryomedicine: Implementation from Nano

to Macroscale
edited by Xiaoming He (The Ohio State University, USA) and
John C. Bischof (University of Minnesota, USA)

Vol. 8 Bioengineering in Wound Healing: A Systems Approach

edited by Martin L. Yarmush (Rutgers University, USA & Harvard Medical
School, USA) and Alexander Goldberg (Tel Aviv University, Israel)

Vol. 9 The World Scientific Encyclopedia of Nanomedicine and Bioengineering II:

Bioimplants, Regenerative Medicine, and Nano-Cancer Diagnosis and
Phototherapy (A 3-Volume Set)
edited by Donglu Shi (University of Cincinnati, USA),
Maoquan Chu (Tongji University, China) and
Jiang Chang (Chinese Academy of Sciences, China)

Forthcoming titles

Vol. 10 Tissue Engineering and Nano Theranostics

edited by Donglu Shi (University of Cincinnati, USA) and
Qing Liu (Tongji University, China)

Vol. 11 Cancer Therapeutics and Imaging:

Molecular and Cellular Engineering and Nanobiomedicine
edited by Kaushal Rege and Sheba Goklany
(Arizona State University, USA)

The complete list of titles in the series can be found at

http://www.worldscientific.com/series/fnbmr

Frontiers in Nanobiomedical Research.indd 1 31-05-17 10:02:36 AM

 THE WORLD SCIENTIFIC
ENCYCLOPEDIA OF NANOMEDICINE
AND BIOENGINEERING II
Bioimplants, Regenerative Medicine, and
Nano-Cancer Diagnosis and Phototherapy
editor-in-chief: Donglu Shi
East Hospital, Tongji University School of Medicine, Shanghai, China
University of Cincinnati, Ohio, USA

 Synthesis and Biomedical Applications

of Graphene Quantum Dots
Vol. 9
editor
Frontiers in
Maoquan Chu Nanobiomedical
Tongji University, China Research

World Scientific
NEW JERSEY • LONDON • SINGAPORE • BEIJING • SHANGHAI • HONG KONG • TA I P E I • CHENNAI • TOKYO

9559_9789814667647_TP_V1.indd 2 14/7/17 9:13 AM

 Published by
World Scientific Publishing Co. Pte. Ltd.
5 Toh Tuck Link, Singapore 596224
USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601
UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

Library of Congress Cataloging-in-Publication Data

Names: Shi, Donglu, editor.
Title: The World Scientific encyclopedia of nanomedicine and bioengineering. II, Bioimplants, regenerative
medicine, and nano-cancer diagnosis and phototherapy / chief editor, Donglu Shi.
Other titles: Encyclopedia of nanomedicine and bioengineering | Bioimplants, regenerative medicine, and
nano-cancer diagnosis and phototherapy | Frontiers in nanobiomedical research ; v. 9. 2251-3965
Description: New Jersey : World Scientific, 2016. | Series: Frontiers in nanobiomedical research ; volume 9 |
Includes bibliographical references and index.
Identifiers: LCCN 2016040312 | ISBN 9789814667586 (hardcover : alk. paper)
Subjects: | MESH: Biocompatible Materials | Nanostructures | Theranostic Nanomedicine |
Tissue Engineering | Prostheses and Implants | Regeneration
Classification: LCC R856.A3 | NLM QT 37 | DDC 610.2803--dc23
LC record available at https://lccn.loc.gov/2016040312

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

Copyright © 2017 by World Scientific Publishing Co. Pte. Ltd.

All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or
mechanical, including photocopying, recording or any information storage and retrieval system now known or to
be invented, without written permission from the publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center,
Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from
the publisher.

Typeset by Stallion Press

Email: [email protected]

Printed in Singapore

JQuek - The World Scientific Encyclopedia of Nanomedicine.indd 2 30-11-16 11:53:23 AM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Preface

Nanobiomedicine refers to the biomedical applications of natural or synthetic

nanomaterials, biological nanodevices or nanomachines. This is an emerging
scientific discipline which has great potential for imaging, early diagnosis, and
targeted therapy of numerous intractable diseases such as cancer. In nanobio-
medicine research field, nanomaterials may be the most important tools
which have greatly improved the advancement of the biomedical areas. The
biomedical applications of nanomaterials include reducing drug resistance,
improving drug water solubility, targeted drug delivery, and controlled drug
release, in vivo tumor non-invasive imaging and targeted cancer therapy.
The most commonly used nanomaterials for biomedical applications
include graphene-based nanoparticles, magnetic nanoparticles, gold
­nanostructures, semiconductor nanoparticles, rare earth doped nanoparticles,
liposomes, polymer micelles, dendrimer-based nanoclusters, etc. Suitable
particle size, morphology and surface zeta potential are important parameters
for these nanoparticles used in biomedical areas. For example, nanoparticles
with small size have the ability of transport through biological barriers and
therefore can deliver the drug to target site. A significant difference between
the tumor and normal tissues is that the tumors exhibit enhanced permeabil-
ity and retention (EPR) effect for macromolecules and nanoparticles. Small
nanoparticles migrating to tumor tissue and finally accumulating in tumor site
after intravenous injection are more easily found than the large nanoparticles.
Therefore, small nanoparticles are usually beneficial for in vivo drug delivery,
­targeted imaging and therapy.
In recent years, nanocomposites containing different types of nanoparticles
have been intensively investigated since those hybrids have multifunctional
properties in biomedical applications. However, the great challenge of those
nanohybrids is that the functions of each nanoparticle may be reduced after
the nanoparticles are incorporated into each other. For example, both the
fluorescent intensity and magnetism of a fluorescent colloid/magnetic crystal

b2227_P2-V1_FM.indd 5 23-Jun-17 2:58:10 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

vi  Preface

hybrid system are lower than those of the individual nanoparticles, and usually
decrease over time. In addition, the sizes of the hybrids significantly increase
compared with those of the individual nanoparticles. Therefore, one type of
nanoparticle exhibiting multifunctional properties may be more useful than the
single system incorporated with different types of nanoparticles.
Semiconductor quantum dot (QD) is one of the good candidates which
meet the above requirements: small size and multifunctional properties.
These nanocrystals are spherical in shape with only 1–10 nm in diameter.
QDs have attracted great interest in biomedical research fields mostly due to
the excellent optical properties such as size-dependent fluorescence, high
fluorescent quantum yield and long-term photostability. They have been
widely applied for in vitro and in vivo fluorescent labeling and imaging. QDs
are also excellent photodynamic therapy (PDT) and photothermal therapy
(PTT) agents which can significantly inhibit mouse tumor growth under red
or near-infrared (NIR) laser irradiation. However, the components of QDs
are mainly from groups II–VI, III–V, or IV–VI, which are composed of toxic
atoms (e.g. cadmium).
Graphene quantum dot (GQD) is another QD which has similar proper-
ties as the semiconductor QD but has no toxic heavy metal atoms. GQDs are
multifunctional nanomaterials with less than 10 nm in size, which are usually
called zero-dimensional (0D) graphene nanosheets.
Both “top-down” and “bottom-up” techniques have been introduced to
prepare such small nanosheets. For example, large-sized graphene can be
physically cut into pieces through strong acid treatment; small aromatic
­molecules after pyrolysis or carbonization can be built into GQDs.
Since GQDs are only several nanometers and have only one or several
layer(s) of carbon atoms, GQDs have huge specific surface areas for loading
drugs. Both the two faces and edge of the GQDs can be loaded with drug
through π–π stacking interaction between the drugs and GQDs and/or
through electrostatic adsorption. Due to the small size, the drug-loaded
GQDs after intravenous injection may conveniently accumulate in tumor
­tissue through EPR effect. Therefore, GQDs may be excellent drug delivery
tools. For the semiconductor QDs, however, they cannot be efficiently
loaded with drugs. As for the large-sized graphene nanosheets, they may not
be suitable for intravenous injection compared with the GQDs.
Although GQDs are small-sized graphene nanosheets, some of their
physical properties are different from those of the large-sized graphene. For
example, large graphene sheets are semimetals or specific semiconductors
with zero bandgap energy. However, when the graphene size decreases to less
than 10 nm (has been changed to GQDs), these small graphene sheets have

b2227_P2-V1_FM.indd 6 23-Jun-17 2:58:10 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Preface vii

remarkable quantum confinement and edge effects, which have great poten-
tial applications in o­ ptical and electronic devices. It has been demonstrated
that GQDs have stable fluorescence ranging from blue to red band. Therefore,
GQDs can be used as fluorescent probes for in vitro labeling and in vivo tar-
geted imaging. The fluorescence of most organic fluorescent dyes and semi-
conductor QDs is not stable in physiological environment, whereas GQDs
usually exhibit resistance to photobleaching in vitro and in vivo, which make
them as potential nanoprobes for long-term tracking the intracellular activi-
ties, biodistribution and metabolism of drugs in vivo.
GQDs are not only electron donors but also electron acceptors.
Therefore, resonance energy transfer (RET) usually occurs between the
GQDs and molecules or particles around the GQDs. Chemical sensor and
biosensor of GQDs have been recently developed based on the fluorescence
RET or luminescence RET. These GQD-based sensors have great potential
for medical, pharmaceutical and food detections.
Upon blue or red laser irradiation, GQDs absorb laser light energy and
produce reactive oxygen species (ROS), especially singlet oxygen (1O2).
Bacterials can be killed by these ROS produced by the laser-triggered GQDs.
Although GQDs have broad light absorption spectrum, the absorption band
mainly locates ranging from ultraviolet to visible wavelength. GQDs under
NIR laser irradiation may therefore produce little ROS. However, PDT in
clinic has been usually applied for the treatment of superficial tumors such as
skin cancer, nasopharyngeal carcinoma and esophageal cancer, and red laser
has been successfully used for irradiating the photosensitizers in tumors.
Therefore, GQDs may have potential for in vivo cancer PDT.
In addition, GQD aqueous solution with a suitable concentration can
convert laser light energy into heat. This means that GQD is also a
­photothermal agent which can be used for cancer PTT. This is an interesting
phenomenon since GQDs are both PDT and PTT agents. The growth of
mouse tumors was significantly inhibited after the tumors were injected with
high-dose GQDs and irradiated with a red (e.g. 671 nm) laser, which may be
due to the synergistic effect of PDT and PTT.
The toxicity of the GQDs to cells and animals may be mainly dependent
on the GQD concentration. Under the same dose, GQDs are safer than other
interesting nanoparticles such as semiconductor QDs and graphene oxide
(GO). For introducing the GQDs into future clinical uses, the in vivo
­metabolic pathways and potential toxicity should be further investigated.
In this book, the synthesis strategies and optical properties of the GQDs,
plasmon behavior in GQDs, GQDs for medical and pharmaceutical analysis
as well as food detections, in vitro and in vivo fluorescent imaging, drug

b2227_P2-V1_FM.indd 7 23-Jun-17 2:58:10 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

viii  Preface

delivery and the toxicity of the GQDs to cells and animals were summarized
and discussed. The front line researchers, who are focusing on the research
of graphene-based nanomaterials and nanobiomedicine, were specially
invited by me, and have written six chapters. I also contributed two chapters
and designed the book cover. I would like to take this opportunity to grate-
fully acknowledge all invited authors for their excellent contributions to this
book. I hope this book will provide useful information on the preparation
and properties as well as the biomedical ­applications of GQDs.

Prof. Maoquan Chu (储茂泉)

Research Center for Translational Medicine at Shanghai East Hospital,
150 Jimo Road, Shanghai 200120, P.R. China
and
School of Life Science and Technology, Tongji University,
1239 Siping Road, Shanghai 200092, P.R. China

b2227_P2-V1_FM.indd 8 23-Jun-17 2:58:10 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

About the Editor

Professor Maoquan Chu was born in Anhui

Province in China. He received his Ph.D. in
Chemical Engineering from East China University
of Science and Technology in 2001. He then did
his postdoctoral work in the School of Life Science
and Technology in Shanghai Jiaotong University,
where he delved into biomaterials and nanotech-
nology. In 2004, he attended the School of Life
Science and Technology at Tongji University. In
2007, he won the China Education Ministry’s
“New Century Excellent Talents Supporting Plan”.
In 2008, he was hired as a Professor and Ph.D. supervisor. He is now a
­principal investigator at the School of Life Science and Technology, Tongji
University and also at the Research Center for Translational Medicine,
Shanghai East Hospital in China. His main area of research is nanobiomedicine,
with a current focus on cancer imaging and therapy using nanobiomaterials.
His recent research work has been published in many important journals such
as Small, Biomaterials, Nanotoxicology, Theranostics, Nanoscale, Carbon, etc.

ix

b2227_P2-V1_FM.indd 9 05-Jul-17 8:53:11 AM

June 16, 2015 14:37 BC: 9335 - Kernel-based Approximation Methods using MATLAB FasshauerMcCourtBook page vi

This page intentionally left blank

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Contents

Prefacev
About the Editorix

Chapter 1 
Properties and Synthesis Strategies of Graphene
Quantum Dots 1
Jiajia Zhang and Hongbin Lu
Chapter 2 
Synthesis and Amino-Functionalization of the Graphene
Quantum Dots 19
Limin Dong and Kejia Wu
Chapter 3 
Plasmons in Graphene Quantum Dots 39
Haifeng Yin
Chapter 4 
Application of Graphene Quantum Dots in Medical
and Pharmaceutical Analyses 57
Xiaolei Zhang, Jing Wang and Gongjun Yang
Chapter 5 Graphene Quantum Dots for Food Analysis 77
Yongkang Ye and Xiaodong Cao
Chapter 6 Fluorescent Graphene Quantum Dots for Bioimaging 97
Shuhua Li, Zetan Fan, Fanglong Yuan and Louzhen Fan
Chapter 7 Graphene Quantum Dots for Drug Delivery 115
Yang Chen and Maoquan Chu
Chapter 8 Toxicity of Graphene Quantum Dots 127
Maoquan Chu

Index139

xi

b2227_P2-V1_FM.indd 11 05-Jul-17 8:53:11 AM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Chapter 1

Properties and Synthesis Strategies

of Graphene Quantum Dots

Jiajia Zhang and Hongbin Lu*

State Key Laboratory of Molecular Engineering of Polymers

Department of Macromolecular Science
Collaborative Innovation Center of Polymer and
Polymer Composite Materials
220 Handan Road, Shanghai, 200433, P.R. China
*[email protected]

Quantum dots (QDs) are usually referred to as semiconductor nanoparticles with their
sizes in the quantum-confined regime, in which the excitons are confined in all the
three spatial dimensions. Typical QDs are inorganic semiconductor nanocrystals from
the group II–VI elements in the periodic table. However, the applications of these QDs
are limited by their internal disadvantages, including intrinsic toxicity (e.g. in the case
of widely studied CdSe QDs) and problems caused by the colloidal stability. Therefore,
developing new QDs and relevant nanomaterials is necessary. Consequently, graphene
quantum dots (GQDs), a class of zero-dimensional (0D) graphitic nanomaterials, have
attracted increasing attention recently. Compared with semiconductor QDs, GQDs
are superior in terms of low cytotoxicity, high dispersity in water and some polarity
organic solvents, resistance to photobleaching and biocompatibility. In addition, the
properties of GQDs are easier to be tuned through surface chemistry, indicating that
researchers can design GQDs with various functionalities for different applications.

1. Introduction
Graphene, a single carbon atom thick carbon film, has attracted strong atten-
tion from both academia and industry due to its extraordinary optical,

b2227_P2-V1_Ch-01.indd 1 23-Jun-17 2:54:08 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

2  J. Zhang & H. Lu

mechanical, thermal and electronic properties combined with large specific

surface areas.1 Pristine graphene has a feature of zero bandgap and cannot yield
fluorescence, which limits its applications in some fields where fluorescence
emission is necessary. Theoretically, the bandgap of graphene can be tuned
from 0 to 6 eV (the bandgap of benzene) by changing its size.2 Thus, convert-
ing two-dimensional (2D) graphene to zero-dimensional (0D) GQDs becomes
an effective approach to expand the application range of graphene.3 GQDs,
also taken as nanosize graphene, exhibit some distinctive properties due to
occurrence of the quantum confinement and edge effects. These unique fea-
tures include small sheet size, tunable photoluminescence (PL), electrochemo-
luminescence (EL), low toxicity, high biocompatibility, chemical inertness,
high stability and ease to be functionalized and so on, which make them
important fluorescent carbon materials and good potential in applications such
as bioimaging, photovoltaic, light-­emitting devices, catalysis, solar cells, sen-
sors, etc.4 Presently, a variety of fabrication methods have been established to
produce GQDs with tunable PL, many intriguing applications have been dem-
onstrated as well. In this chapter, we summarize the properties and synthesis
strategies of GQDs, and analyze the possible challenges in related fields.

2. Characterization and Properties of GQDs

GQDs and semiconductor quantum dots (QDs) show obvious similarities
such as the ­electrons in them are confined in all the three spacial dimensions.
The energy bandgaps of electrons are dependent on the sizes, which means
the fluorescent properties can be tuned by adjusting the sizes. In fact, the
size-dependent fluorescence of GQDs is less prominent compared with that
of semiconductor QDs because various defects, such as edge states, doped
heteroatoms, surface functional groups, may also derive PL emissions in
GQDs.

2.1. Characterization of GQDs

Besides GQDs, other nanosize PL carbon materials have been reported,
including carbon nanodots and polymer dots.5 These three kinds of carbon
materials have at least one dimension less than 10 nm in size, and tunable PL
properties. Although they exhibit similar properties to some extent, their inter-
nal structures are quite different. Thus, it is necessary to distinguish GQDs
from other PL carbon materials, which enables us to elucidate their intrinsic
luminescence mechanisms. Different from semiconductor QDs and carbon
nanodots, which have quasi-spherical structures with diameter smaller than 10

b2227_P2-V1_Ch-01.indd 2 23-Jun-17 2:54:08 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Properties and Synthesis Strategies of Graphene Quantum Dots 3

nm, graphene QDs possess lamellar morphology similar to single- or few-lay-

ered graphene. The lateral size of GQDs ranges from several nanometers to 60
nanometers with a thickness of <3 nm in most cases.6 Experimentally, the size
of GQDs can be characterized by transmission electron microscopy (TEM)
while the thickness is usually measured by atomic force microscopy (AFM)
(Figures 1(A) and 1(B)).7 In high resolution transmission electron microscopy

Figure 1. (A) and (B) TEM and AFM images of GQDs. Reprinted with permission from
Ref. [21]. Copyright (2012) American Chemical Society. (C), (D) and (F) Raman, XRD and
XPS spectra of GQDs. Reprinted with permission from Ref. [8]. Copyright (2012) American
Chemical Society. (E) FTIR spectra of GQDs. Reprinted with permission from Ref. [9].
Copyright (2012) WILEY-VCH Verlag GmbH & Co.KGaA. (G) MALDI–TOF mass spec-
trum of GQDs. Reprinted with permission from Ref. [10]. Copyright (2010) American
Chemical Society.

b2227_P2-V1_Ch-01.indd 3 23-Jun-17 2:54:08 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

4  J. Zhang & H. Lu

(HR-TEM) images, both (100) in-plane lattice spacing and (002) interlayer
spacing co-exist.6
X-ray diffraction (XRD) is also used to characterize the crystal structure
of GQDs.8 In most cases, the XRD patterns of GQDs show a broad peak with
an interlayer space larger than 0.34 nm (the interlayer space of graphite)
(Figure 1(D)). The broad diffraction peaks arise from the small size effect of
GQDs whereas the increased interlayer space primarily originates from the
introduction of oxygen-containing functional groups and doped heter-
oatoms. Raman spectroscopy is another powerful, non-destructive tool to
characterize GQDs.8 The Raman spectrum of GQDs shows two peaks
centered around 1360 and 1580 cm–1 (Figure 1(C)), corresponding to
­
D band and G band, respectively. The D band results from the breathing
mode of the sp2 carbon atoms, which is caused by defects such as functional
groups, doped heteroatoms and structural disorders. The G band is the
result of first-order scattering of the E2g mode, corresponding to the in-plane
vibration of sp2 hybridized C–C bonds.
Fourier transform infrared (FTIR)9 and X-ray photoelectron spectroscopy
(XPS) techniques8 are commonly employed to analyze the chemical compo-
nent of GQDs (Figures 1(E) and 1(F)). Matrix-assisted laser desorption/
ionization time of flight mass spectrometry (MALDI–TOF-MS) is thought
to be the best way to survey GQDs on an ensemble level (Figure 1(G)).10 The
spectra of MALDI–TOF-MS can yield reasonable, yet no quantitative,
­estimation for the chemical composition of GQDs, which can be taken as a
supplement of FTIR and XPS.

2.2. Properties of GQDs

GQDs have optical absorption properties similar to that of graphene oxide
(GO).2 The ultraviolet–visible (UV–vis) spectrum of GQDs shows two char-
acteristic peaks: a main peak in the UV region of 260–320 nm and a shoulder
peak in the range of 270–390 nm with a long tail extending to visible region
(Figure 2(A)). The main peak arises from the π–π* transition of C=C bonds
while the shoulder is the result of n–π* transition of C=O bonds. The optical
absorption spectra of GQDs obtained by different synthesis methods are dis-
tinctive. The size, shape, and surface functional groups play a crucial role in
their absorption property. Doping heteroatoms in the in-plane lattice of
GQDs and post-modification of the surface chemical components can alter
these two characteristic peaks. In addition, solvents11 and temperature12 may
impact them to some extent.
Fluorescence is the most attracting property of GQDs. GQDs exhibit
tunable fluorescence, high fluorescent stability, high quantum yield and

b2227_P2-V1_Ch-01.indd 4 23-Jun-17 2:54:09 PM

b2227_P2-V1_Ch-01.indd 5

b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Properties and Synthesis Strategies of Graphene Quantum Dots
Figure 2. (A) The absorbance and excitation spectra of the GQDs solution. Reprinted with permission from Ref. [16]. Copyright (2010)
WILEY-VCH Verlag GmbH & Co.KGaA. (B) The steady state emission spectrum of GQDs and fluorescence lifetime distribution measured using
TCSPC at the emission intensity peak (λ = - 430 nm). Reprinted with permission from Ref. [9]. Copyright (2012) WILEY-VCH Verlag GmbH
& Co.KGaA. (C) PL spectra of GQD solution under different excitation wavelength. Reprinted with permission from Ref. [17]. Copyright
23-Jun-17 2:54:09 PM

5
(2014) WILEY-VCH Verlag GmbH & Co.KGaA. (D) Mechanism for the broadband emission in the N-GQDs. Reprinted with permission from
Ref. [18]. Copyright (2013) Nature Publishing Group.
 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

6  J. Zhang & H. Lu

unique fluorescence quenching feature.5–7 This makes it an appealing candi-

date for many applications, for example, bioimaging, photovoltaic, sensor,
clinical diagnostic, etc.
GQDs can emit different colors of PL, from deep UV to red region,13
depending on the synthesis method and post-modification processes.
Among them, blue and green are the two most common colors. Usually, the
size and chemical composition of GQDs are inhomogeneous due to
uncontrollable synthesis processes.6 For this reason, the emission spectra of
GQDs are broad and depend on the excitation wavelength, implying that
the PL peaks of GQDs generate red-shift with increasing excitation
wavelengths (Figure 2(C)).9,14
Compared to organic fluorescent molecules, one of the advantages of
GQDs is their outstanding optical stability, which is important to those long-
term real-time imaging operations.5 Most GQDs can emit bright fluorescence
even after long time exposure under UV light. GQDs fabricated by hydro-
thermal treatment of trinitropyrene even exhibit PL.15 Furthermore, the
intensity and brightness of photoluminescence are markedly enhanced upon
3 h continuous exposure under 100 W xenon lamp. In contrast, semiconduc-
tor QDs usually show a typical photobleaching effect under the same
condition.
In addition, up-conversion PL of GQDs has been reported by several
groups.19–21 This phenomenon is attributed to a multi-photon activation pro-
cess or an anti-Stokes transition. This phenomenon may expand the applica-
tions of GQDs to bioscience and energy fields. Nevertheless, some researchers
argued that the up-conversion PL observed might be artificial, rather than
from GQDs themselves.22,23 It is demonstrated that the second-order diffrac-
tion light of Xenon lamp at l/2 coexisting in the excitation light actually may
contribute to the observed up-conversion PL. In this point, the second order
grating diffraction would be ruled out when considering the up-conversion
phenomenon.
Until now, the PL mechanism of GQDs is still open,24,25 mainly due to
the difficulty of controlled synthesis. Although full understanding of the PL
mechanism of GQDs remains challenging at this time, the spectral features
of the emission and the related structural characteristics can supply valuable
information. Generally, the PL of GQDs could be divided into two kinds.
One of them arises from the quantum confinement effect, typically relating
to their lateral sizes and edge state (Figure 2(D)).26–29 The PL of GQDs
caused by the quantum confinement effect has been proved by both theo-
retical calculations and experimental observation. With the increase of the
size of GQDs, the absorption peak energy decreases. The other relates to

b2227_P2-V1_Ch-01.indd 6 23-Jun-17 2:54:09 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Properties and Synthesis Strategies of Graphene Quantum Dots 7

defects involving functional groups, doped heteroatoms and edge

structures.30–35

3. Synthesis of GQDs
Similar to graphene, the synthetic strategies of GQDs can be divided into two
categories: top-down strategy and bottom-up strategy.36 The PL behavior of
GQDs depends on the synthesis strategies. To alter the fluorescence proper-
ties of GQDs and enhance their quantum yield, post-modification such as
chemical doping and surface functionalization is usually performed. In the
following, we summarize the synthesis and modification strategies of GQDs
with emphasis on the reaction mechanisms. Considering the difficulty in
revealing the internal mechanisms of GQDs, we highlight stepwise organic
synthesis strategy because well-defined GQDs can be prepared by this
method, which is important to reveal the origin of PL of GQDs and unravel
new properties and applications.

3.1. Top-down strategies

The top-down strategy involves cutting carbonaceous materials containing
graphene sheets into smaller nanosheets through chemical and physical
approaches, including hydrothermal,16 solvothermal,37 ultrasonic,17 micro-
wave13 and electrochemical treatment, electron-beam lithography, pulsed
laser.38 The carbonaceous materials may be carbon fibers,7 graphene,30 GO,39
graphite,37,40–43,44 carbon black,45 carbon nanotubes,46,47 coal.48 Here, we sum-
marize the top-down synthesis of GQDs and the related cutting mechanisms.
Electron-beam lithography. GQDs were prepared first by electron-beam
lithography in 2008. After graphene was peeled off from graphite by mechan-
ical exfoliation, which was then deposited onto the n-Si substrate that was
coated with 300 nm SiO2 and 90 nm poly(methyl methacrylate) (PMMA) as
a protective layer. Subsequently, Ar/O2 (9:1) plasma as reactive ion etchant
(RIE) was introduced to eliminate unprotected graphene. After removing the
residual PMMA, single GQDs with the thickness of about 0.5 nm was
obtained. For these GQDs, clear and reproducible Coulomb resonances were
observed. The charging energy of GQDs was estimated to be about 3.5 meV
from the Coulomb diamond. Although this method could be difficult to
obtain smaller GQDs, which is necessary for the research of the quantum
states, it paves a way for further studies on GQDs.
Hydrothermal treatment. Hydrothermal treatment is the earliest chemical
method reported for preparing GQDs with the size down below 10 nm by

b2227_P2-V1_Ch-01.indd 7 23-Jun-17 2:54:09 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

8  J. Zhang & H. Lu

cutting oxidized graphene sheets into nanosize GQDs (Figure 2(A)).16 In this
method, graphite oxide was prepared first by Hummers or modified Hummers
method. The graphite oxide obtained was thermally deoxidized in tube fur-
nace to give graphene sheets (GSs). Subsequently, the GSs were re-oxidized in
the mixture of H2SO4 and HNO3 (volume ratio = 1:3) under mild sonication
and the oxidized GSs were dispersed in water to form a pH = 8 suspension.
The solution was then treated at 200°C for a period of time to yield GQDs.
Fluorescence spectra prove that the as-synthesized GQDs emit bright blue
fluorescence even in the neutral solution. The oxidized GSs are considered to
have more carbonyl groups rather than epoxy groups, as shown in FTIR spec-
tra in which the oxidized GSs exhibit a strong carbonyl signal and a weak epoxy
signal. The cutting mechanism was presented on the basis of the above results,
namely, the presence of the linear defects makes the oxidized GSs easier to be
attacked under hydrothermal condition. Besides, the portion of ultrafine pieces
surrounded by epoxy lines and edges are further broken down when the bridg-
ing oxygen atoms in epoxy chains are removed. This results in the formation
of GQDs. In addition, it is likewise possible that the fluorescence-emitting
might arise from free zigzag sites in a carbene-like triplet ground state. This
mechanism is consistent with the pH-dependent fluorescence property, that is,
the GQDs emit bright fluorescence under alkaline and neutral conditions, but
under acidic conditions, the fluorescence is nearly completely quenched.
Modified hydrothermal treatment. After the above seminal work, many
further studies regarding hydrothermal synthesis of GQDs through top-down
strategy have been published.48 In view of the fact that the GQDs fabricated
in the previous work are highly disordered, Pan et al. improved their cutting
route. They employed the high-temperature thermally reduced graphite
oxide as the precursor and then treated it under strongly alkaline condition.
The resulting GQDs are very small, with a lateral size of about 3 nm, well
crystallized. They emit bright, stable green fluorescence, and have good
reversibility under different pH conditions. Despite the difference in crystal-
linity, however, the fluorescence emitting of these GQDs is not tunable. Thus,
Tetsuka et al.49 designed another hydrothermal approach to synthesize GQDs
with edge-terminated primary amines. In this work, the GO without thermal
reduction was used as the precursor and hydrothermal treatment was per-
formed in ammonia solution. The ammonia can react with the epoxy group
on GO sheets by nucleophilic substitution, which can not only extract sp2
domains through the ring-opening reaction of epoxy groups but also decorate
the edge of GQDs due to the bonding of amines. The fluorescence of the
extracted GQDs can be simply controlled by adjusting the temperature of
hydrothermal treatment and the concentration of ammonia. As a result, PL
can be tuned from violet to yellow. Ab initio calculations were used to

b2227_P2-V1_Ch-01.indd 8 23-Jun-17 2:54:09 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Properties and Synthesis Strategies of Graphene Quantum Dots 9

elucidate how primary amines influence the electronic structure of GQDs.

The results indicate that the primary amines on the edges lift one of the
degenerate in the highest occupied molecular orbital (HUMO) to a higher
energy because of the strong orbital interaction with the –NH2 group. The
bandgap between the HUMO and the lowest unoccupied molecular orbital
(LUMO) decreases with increasing amounts of the primary amine, leading to
the occurrence of tunable PL.
Solvothermal treatment. Similar to hydrothermal treatment, solvothermal
method was also used to prepare GQDs.50 Zhu et al. implemented a one-step
solvothermal approach to synthesize GQDs from GO. The GQDs emit visi-
ble green fluorescence under UV light. An interesting phenomenon is that
the fluorescence intensity keeps constant in the pH range of 4–8 but decreases
in higher or lower pH solutions. It is suggested that the fluorescence is
related to both the quantum size effect and the surface environment, which
is consistent with the FTIR results.
Chemical oxidation. Chemical oxidation is widely used to synthesize
GQDs (Figure 3(D)). This method highly depends on different carbon
sources, such as carbon fibers,47 coal,18 carbon nanotubes,46 graphite41 and
carbon black45 in the mixture of strong acids (sulfuric acid and nitric acid).
The carbon sources typically contain graphene sheets. The oxygen-containing
groups such as C=O, C–O, O–H are introduced to the surface of GQDs dur-
ing the oxidation. This method provides a simple approach to synthesize
GQDs using cheap, abundant precursors. The fluorescence of synthesized
products can also be tuned by changing reaction temperature and time.
Electrochemical cleavage. GQDs have also been prepared by electro-
chemical cleavage of precursors (Figure 3(C))17,40 such as carbon nanotubes,
graphite, reduced GO. It is believed that OH• and O• generated in the
anodic oxidation of water act as “scissors” to cut precursors into GQDs.
Oxygen-containing groups or heteroatoms are typically introduced to the
resulting GQDs. Heteroatoms doping in GQDs can be achieved by a pre-
cursors approach, that is, heteroatom-containing precursors were used as
carbon sources. For example, Li et al. produced N-doped GQDs by such a
simple, effective electrochemical strategy. Unlike the N-free counterparts,
N-doped GQDs exhibit a superior electrocatalytic activity for the oxygen
reduction reaction in alkaline solutions.
Microwave irradiation. Microwave irradiation has been widely used for
material synthesis (Figure 3(B)).9 Compared to traditional heating tech-
niques, microwave irradiation can heat reaction media in a fast, uniform man-
ner, contributing to shorten the reaction time, enhance the yield and purity
of products. For the same purpose, microwave is also used to synthesize
GQDs from graphite or GO. The reaction media are usually prepared by

b2227_P2-V1_Ch-01.indd 9 23-Jun-17 2:54:09 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

10  J. Zhang & H. Lu

Figure 3. (A) Hydrothermal synthesis of GQDs from oxidized graphene sheets. Reprinted
with permission from Ref. [16]. Copyright (2010) WILEY-VCH Verlag GmbH & Co.KGaA.
(B) Microwave synthesis of GQDs from GO. Reprinted with permission from Ref. [9].
Copyright (2012) WILEY-VCH Verlag GmbH & Co.KGaA. (C) Electrochemistry synthesis
of GQDs from 3D graphene. Reprinted with permission from Ref. [17]. Copyright (2014)
WILEY-VCH Verlag GmbH & Co.KGaA. (D) Oxidation synthesis of GQDs from coal.
Reprinted with permission from Ref. [18]. Copyright (2013) Nature Publishing Group.

b2227_P2-V1_Ch-01.indd 10 23-Jun-17 2:54:10 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Properties and Synthesis Strategies of Graphene Quantum Dots 11

dissolving strong oxidants (such as nitric acid and potassium permanganate)

with sulfuric acid. During the microwave treatment, the oxidation cleavage of
precursors occurs quickly. The resulting GQDs usually have more uniform
size distribution compared with those products obtained through other
routes.
Fenton’s reaction. From the point of view of chemistry, GO can be
regarded as a 2D aromatic macromolecule in which part of its carbon atoms
connected with oxygen-containing functional groups.51 Under the UV
­irradiation, Fenton reagent can generate a large amount of peroxide and
hydroxyl radical to attack the carbon atoms connected with oxygenous
groups, resulting in the cleavage of GO. This enables GO to be cut into
porous ­graphene and GQDs in the early stage. The obtained porous GO is
further cut to GQDs in the subsequent stage. This reaction proceeds rapidly
and also provides an effective route to prepare porous graphene sheets.

3.2. Bottom-up strategies

The bottom-up strategy is mainly based on the direct pyrolysis or carboniza-
tion of organic precursors during hydrothermal, electrochemical treatments,
and microwave radiation.52–57
Compared with the top-down methods, the bottom-up strategy has advan-
tages in modulating the chemical composition and PL features because organic
precursors and reaction conditions can be adjusted and controlled conveniently.
Self-passivated GQDs can be prepared by microwave treatment of glucose
aqueous solutions (Figure 4(A)). Using some carbon sources having nitrogen-
containing species, N-doped GQDs with various lateral sizes have been pre-
pared. One of the outstanding advantages of this strategy lies in the fact that
the starting raw materials can be extended to quite a few small molecules con-
taining both carbon and nitrogen elements. N-doped GQDs exhibit a broad
emission range, from 300 to 1000 nm or more, which can be prepared by one-
pot microwave heating method when glucose is used as carbon source and
ammonia as nitrogen source. Ammonia acts as both catalyst and doping agent
in this process. Actually, the doping atoms are not limited to nitrogen, other
atoms such as sulfur, boron, phosphorus and fluorine can also be doped into
the lattice of GQDs. Heating small molecules beyond their melting point can
also generate GQDs. Considering that GQDs usually have polycrystalline or
highly defective structures, which limit the applications of GQDs in fields such
as opto-electronics and clinical diagnostics, Wang et al. reported a large-scale
bottom-up strategy to synthesize single-crystalline GQDs.15 This method
involves the nitration of pyrene followed by hydrothermal treatment in

b2227_P2-V1_Ch-01.indd 11 23-Jun-17 2:54:10 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

12  J. Zhang & H. Lu

Figure 4. (A) Microwave-assisted hydrothermal treatment of glucose. Reprinted with

permission from Ref. [13]. Copyright (2012) American Chemical Society. (B) Synthesis of
GQDs by age-opening buckminsterfullerene. Reprinted with permission from Ref. [57].
Copyright (2015) WILEY-VCH Verlag GmbH & Co.KGaA. (C) Stepwise synthesis of
GQDs. Reprinted with permission from Ref. [59]. Copyright (2010) American Chemical
Society. (D) Synthesis of single-crystalline GQDs from pyrene. Reprinted with permission
from Ref. [15]. Copyright (2014) Nature Publishing Group.

different alkaline aqueous solutions (Figure 4(D)). High quality GQDs can also
be synthesized by rupturing C60 molecules (Figure 4(B)).57,58
Formation mechanisms. Although considerable efforts have been con-
ducted in the synthesis of GQDs via pyrolysis or carbonization of organic pre-
cursors, few efforts were dedicated to the study of the formation mechanisms.
Dong et al.52 attributed the formation of GQDs to the carbonization of citric

b2227_P2-V1_Ch-01.indd 12 23-Jun-17 2:54:11 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Properties and Synthesis Strategies of Graphene Quantum Dots 13

acid based on the observation in which the GO grows with extended reaction
times. Tang et al. proposed another different mechanism. Glucose is first dehy-
drated to form the nucleus composed of sp2 carbon atoms, followed by the edge
growth of GQDs. The glucose molecules generate new sp2 ­carbon by dehydra-
tion at the edge of GQDs, resulting in increasing lateral sizes in the extended
reaction time. Nevertheless, this hypothesis could work only for the specific
process. To further elucidate the formation mechanism, effectively monitoring
the whole reaction process is of great importance. Several points need to be
considered in this regard. (1) How does the transformation of carbon from sp3
to sp2 hybridization occur? (2) How do the functional groups in the raw mate-
rial molecules impact the formation process? (3) How does the re-organization
of the species take place? (4) Whether or not the pyrolysis or carbonization of
different organic precursors has something in common? Apparently, to answer
these questions, there is still a large amount of work to be conducted.

3.3. Stepwise organic synthesis of GQDs

To reveal the exact fluorescence mechanism of GQDs, synthesis of GQDs with
well-defined structures is an important prerequisite. Well-defined GQDs are
model systems that help to clarify the optical mechanisms of carbon materials.
Stepwise organic synthesis strategy provides an opportunity to achieve this
goal since well-controlled GQDs with an atom precision can be prepared by
this method.59,69 Stabilization of GQDs is a critical challenge during the syn-
thesis due to the decreasing solubility as their size increases. Li et al.59 resolved
this problem by introducing a 3D “cage” around GQDs, which enlarges the
distance between GQDs and impairs the short-range interaction (Figure
4(C)). This was achieved by covalently attaching multiple 2′,4′,6′-tralkylphe-
nyl units to the edge of GQDs. Using this method, they synthesized stable
GQDs containing 132, 168, and 170 conjugated carbon atoms. After fusing
the phenyl moieties, purified GQDs were obtained. The bandgap and redox
potential of GQDs can be adjusted independently by changing size and func-
tional groups, respectively. This is helpful for further developing opto-electri-
cal applications and also provides a simple route to predict the bandgap and
redox potentials of GQDs. These progresses in controllable synthesis of
GQDs are impressive, however, some challenges still exist. At present, the
functional groups and doped heteroatoms in GQDs still lack effective control
methods. In addition, due to the complexity and inhomogeneity of GQDs,
mechanistic studies of many applications, including catalysis, sensor and fuel
cells, remain quite difficult at the present stage. By accurately controlling the
chemical environment of heteroatoms (e.g. nitrogen, boron, sulfur, phospho-
rus, fluorine) and the surface functional groups, the exact optical, catalytic and

b2227_P2-V1_Ch-01.indd 13 23-Jun-17 2:54:11 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

14  J. Zhang & H. Lu

other related mechanisms can be further understood, which is important for

fundamental research involving GQDs, and will offer more opportunities to
uncover new phenomena and applications as well.

3.4. Post-modification of GQDs

As aforementioned, doped heteroatoms and surface functional groups play
critical roles in the properties and applications of GQDs. They can be achieved
by one-step synthesis in some cases, but in other cases, they could be difficult
to accomplish by this strategy. In this regard, post-modification is an alterna-
tive approach, in which GQDs can be modified by post-processing.58,60–68
Post-modification allows one to design GQDs with rationally improved prop-
erties. Polymer passivation is an often-adopted method to improve the quan-
tum yield of GQDs. Polyethylene glycol (PEG) is the most used polymer for
surface decoration of GQDs. Novak et al. have decorated GQDs with various
molecular weights of PEG. Low molecule weight PEG modified GQDs
exhibit better interaction with the active layer, leading to a 36% improvement
in conversion efficiency of solar cells. The enhancement mechanism was
believed to originate from the faster P3HT exciton dissociation in GQDs-
containing cells. Luo et al. have reported a diazonium chemistry method to
modulate the edge of GQDs with different aryl groups. The attachment of
aryl groups can protect the PL active sites and introduce the interaction
between GQDs and aryl groups. Meanwhile, this method tunes the PL prop-
erties of GQDs, and improves their ­quantum yield. Interestingly, the aryl-
modified GQDs also showed good pH tolerance. They can emit bright
fluorescence even in acidic media, which is valuable for those applications in
acidic environments such as bioimaging in human body, where the pH value
of solutions could be as low as 1 (e.g. in stomach).

4. Summary and Outlook

We summarize the properties and synthesis strategies of GQDs, including
related fundamental understandings. In spite of significant progress in this
regard, there still exist some critical challenges that need to be overcome. To
begin with, the strategy for controlled synthesis of GQDs still needs to be
improved, especially for controlled chemical doping and surface functionali-
zation of GQDs. In addition, the formation mechanisms of the bottom-up
synthesis are still unclear. Further efforts are necessary for clarify the underly-
ing mechanism of these complicated processes, including pyrolysis, transfor-
mation from sp3-carbon to sp2-carbon and re-organization of species during
the GQD formation, etc. Meanwhile, the PL mechanisms are still under

b2227_P2-V1_Ch-01.indd 14 23-Jun-17 2:54:11 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Properties and Synthesis Strategies of Graphene Quantum Dots 15

debate, which inhibits to some extent the exploitation of GQDs with new
properties and applications. Apparently, the research for GQDs is presently
still at its early stage and more systematic efforts will largely boost their fur-
ther development and practical applications.

References
1. Zhu YW, Murali S, Cai WW, Li XS, Suk JW, Potts JR, Ruoff RS. Graphene and graphene
oxide: Synthesis, properties, and applications. Adv Mater 2010; 22(35): 3906–3924.
2. Eda G, Lin YY, Mattevi C, Yamaguchi H, Chen HA, Chen IS, Chen CW, Chhowalla M.
Blue photoluminescence from chemically derived graphene oxide. Adv Mater 2010;
22(4): 505–509.
3. Cao L, Meziani MJ, Sahu S, Sun YP. Photoluminescence properties of graphene versus
other carbon nanomaterials. Accounts Chem Res 2013; 46(1): 171–180.
4. Shen JS, Zhu YH, Yang XL, Li CZ. Graphene quantum dots: Emergent nanolights for
bioimaging, sensors, catalysis and photovoltaic devices. Chem Commun 2012; 48(31):
3686–3699.
5. Zhu SN, Song YB, Zhao XH, Shao JR, Zhang JH, Yang B. The photoluminescence
mechanism in carbon dots (graphene quantum dots, carbon nanodots, and polymer dots):
Current state and future perspective. Nano Res 2015; 8(2): 355–381.
6. Zheng XT, Ananthanarayanan A, Luo KQ, Chen P. Glowing graphene quantum dots and
carbon dots: Properties, syntheses, and biological applications. Small 2015; 11(14):
1620–1636.
7. Peng J, Gao W, Gupta BK, Liu Z, Romero-Aburto R, Ge L, Song L, Alemany LB, Zhan
X, Gao G, et al. Graphene quantum dots derived from carbon fibers. Nano Lett 2012;
12(2): 844–849.
8. Li Y, Zhao Y, Cheng H, Hu Y, Shi G, Dai L, Qu L. Nitrogen-doped graphene quantum
dots with oxygen-rich functional groups. J Am Chem Soc 2012; 134(1): 15–18.
9. Li L-L, Ji J, Fei R, Wang C-Z, Lu Q, Zhang J-R, Jiang L-P, Zhu J-J. A Facile Microwave
Avenue to electrochemiluminescent two-color graphene quantum dots. Adv Funct Mater
2012; 22(14): 2971–2979.
10. Yan X, CuX, Li BS. Large, solution-processable graphene quantum dots as light absorbers
for photovoltaics. Nano Lett 2010; 10(5): 1869–1873.
11. Colherinhas G, Fileti EE, Chaban VV. Can inorganic salts tune electronic properties of
graphene quantum dots? Phys Chem Chem Phys 2015; 17(26): 17413–17420.
12. Li C, Yue Y. Fluorescence spectroscopy of graphene quantum dots: Temperature effect at
different excitation wavelengths. Nanotechnology 2014; 25(43): 435703.
13. Tang L, Ji R, Cao X, Lin J, Jiang H, Li X, Teng KS, Luk CM, Zeng S, Hao J, et al. Deep
ultraviolet photoluminescence of water-soluble self-passivated graphene quantum dots.
ACS Nano 2012; 6(6): 5102–5110.
14. Deng X, Sun J, Yang S, Shen H, Zhou W, Lu J, Ding G, Wang Z. The emission wave-
length dependent photoluminescence lifetime of the N-doped graphene quantum dots.
Applied Phys Lett 2015; 107(24): 241905.
15. Wang L, Wang Y, Xu T, Liao H, Yao C, Liu Y, Li Z, Chen Z, Pan D, Sun L, et al. Gram-
scale synthesis of single-crystalline graphene quantum dots with superior optical proper-
ties. Nat Commun 2014; 5: 5357.

b2227_P2-V1_Ch-01.indd 15 23-Jun-17 2:54:11 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

16  J. Zhang & H. Lu

16. Pan D, Zhang JC, Li Z, Wu MH. Hydrothermal route for cutting graphene sheets into
blue-luminescent graphene quantum dots. Adv Mat 2010; 22(6): 734–738.
17. Ananthanarayanan A, Wang X, Routh P, Sana B, Lim S, Kim D-H, Lim K-H, Li J, Chen P.
Facile Synthesis of graphene quantum dots from 3D graphene and their application for
Fe3+ sensing. Adv Funct Mater 2014; 24(20): 3021–3026.
18. Ye R, Xiang C, Lin J, Peng Z, Huang K, Yan Z, Cook NP, Samuel EL, Hwang CC, Ruan G,
et al. Coal as an abundant source of graphene quantum dots. Nat Commun 2013; 4: 2943.
19. Shen J, Zhu Y, Chen C, Yang X, Li C. Facile preparation and upconversion luminescence
of graphene quantum dots. Chem Commun 2011; 47(9): 2580–2582.
20. Li M, Wu W, Ren W, Cheng H-M, Tang N, Zhong W, Du Y. Synthesis and upconversion
luminescence of N-doped graphene quantum dots. Appl Phys Lett 2012; 101(10): 103107.
21. Zhuo S, Shao M, Lee S-T. Upconversion and downconversion fluorescent graphene quan-
tum dots: Ultrasonic preparation and photocatalysis. ACS Nano 2012; 6(2): 1059–1064.
22. Gan Z, Wu X, Zhou G, Shen J, Chu PK. Is there real upconversion photoluminescence
from graphene quantum dots? Adv Opt Mate 2013; 1(8): 554–558.
23. Wen X, Yu P, Toh YR, Ma X, Tang J. On the upconversion fluorescence in carbon nano-
dots and graphene quantum dots. Chem Commun 2014; 50(36): 4703–4706.
24. Volk C, Neumann C, Kazarski S, Fringes S, Engels S, Haupt F, Muller A, Stampfer C.
Probing relaxation times in graphene quantum dots. Nat Commun 2013; 4: 1753.
25. Röding M, Bradley SJ, Nydén M, Nann T. Fluorescence lifetime analysis of graphene
quantum dots. J Phys Chem C 2014; 118(51): 30282–30290.
26. Tang L, Ji R, Li X, Teng KS, Lau SP. Size-dependent structural and optical characteristics
of glucose-derived graphene quantum dots. Part Part Systs Char 2013; 30(6): 523–531.
27. Kim S, Hwang SW, Kim M-K, Shin DY, Shin DH, Kim CO, Yang SB, Park JH, Hwang
E, Choi S-H, et al. Anomalous behaviors of visible luminescence from graphene quantum
dots: Interplay between size and shape. ACS Nano 2012; 6(9): 8203–8208.
28. Lingam K, Podila R, Qian H, Serkiz S, Rao AM. Evidence for edge-state photoluminescence
in graphene quantum dots. Adv Funct Mater 2013; 23(40): 5062–5065.
29. Ritter KA, Lyding JW. The influence of edge structure on the electronic properties of
graphene quantum dots and nanoribbons. Nat mater 2009; 8(3): 235–242.
30. Wang S, Cole IS, Zhao D, Li Q. The dual roles of functional groups in the photolumi-
nescence of graphene quantum dots. Nanoscale 2016; 8(14): 7449–7458.
31. Tam TV, Trung NB, Kim HR, Chung JS, Choi WM. One-pot synthesis of N-doped gra-
phene quantum dots as a fluorescent sensing platform for Fe3+ ions detection. Sensor
Actuat B-Chem 2014; 202: 568–573.
32. Sk MA, Ananthanarayanan A, Huang L, Lim KH, Chen P. Revealing the tunable photo-
luminescence properties of graphene quantum dots. J Mater Chem 2014; 2(34): 6954.
33. Permatasari FA, Aimon AH, Iskandar F, Ogi T, Okuyama K. Role of C-N configurations
in the photoluminescence of graphene quantum dots synthesized by a hydrothermal
route. Sci Rep 2016; 6: 21042.
34. Liu F, Jang MH, Ha HD, Kim JH, Cho YH, Seo TS. Facile synthetic method for pristine
graphene quantum dots and graphene oxide quantum dots: Origin of blue and green
luminescence. Adv Mater 2013; 25(27): 3657–3662.
35. Kumar GS, Roy R, Sen D, Ghorai UK, Thapa R, Mazumder N, Saha S, Chattopadhyay
KK. Amino-functionalized graphene quantum dots: Origin of tunable heterogeneous
photoluminescence. Nanoscale 2014; 6(6): 3384–3391.
36. Lin LP, Rong MC, Luo F, Chen DM, Wang YR, Chen X. Luminescent graphene quan-
tum dots as new fluorescent materials for environmental and biological applications. Trend
Anal Chem 2014; 54: 83–102.

b2227_P2-V1_Ch-01.indd 16 23-Jun-17 2:54:11 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Properties and Synthesis Strategies of Graphene Quantum Dots 17

37. Kwon W, Kim YH, Lee CL, Lee M, Choi HC, Lee TW, Rhee SW. Electroluminescence
from graphene quantum dots prepared by amidative cutting of tattered graphite. Nano
Lett 2014; 14(3): 1306–1311.
38. Habiba K, Makarov VI, Avalos J, Guinel MJF, Weiner BR, Morell G. Luminescent gra-
phene quantum dots fabricated by pulsed laser synthesis. Carbon 2013; 64: 341–350.
39. Zhang M, Bai L, Shang W, Xie W, Ma H, Fu Y, Fang D, Sun H, Fan L, Han M, et al.
Facile synthesis of water-soluble, highly fluorescent graphene quantum dots as a robust
biological label for stem cells. J Mater Chem 2012; 22(15): 7461.
40. Zhu Y, Wang G, Jiang H, Chen L, Zhang X. One-step ultrasonic synthesis of graphene
quantum dots with high quantum yield and their application in sensing alkaline phos-
phatase. Chem Commun 2015; 51(5): 948–951.
41. Shin Y, Lee J, Yang J, Park J, Lee K, Kim S, Park Y, Lee H. Mass production of graphene
quantum dots by one-pot synthesis directly from graphite in high yield. Small 2014;
10(5): 866–870.
42. Shin Y, Park J, Hyun D, Yang J, Lee JH, Kim JH, Lee H. Acid-free and oxone oxidant-
assisted solvothermal synthesis of graphene quantum dots using various natural carbon
materials as resources. Nanoscale 2015; 7(13): 5633–5637.
43. Lin L, Zhang S. Creating high yield water soluble luminescent graphene quantum dots
via exfoliating and disintegrating carbon nanotubes and graphite flakes. Chem Commun
2012; 48(82): 10177–10179.
44. Sun Y, Wang S, Li C, Luo P, Tao L, Wei Y, Shi G. Large scale preparation of graphene
quantum dots from graphite with tunable fluorescence properties. Phys Chem Chem Phys
2013; 15(24): 9907–9913.
45. Dong Y, Chen C, Zheng X, Gao L, Cui Z, Yang H, Guo C, Chi Y, Li CM. One-step and
high yield simultaneous preparation of single- and multi-layer graphene quantum dots
from CX-72 carbon black. J Mater Chem 2012; 22(18): 8764.
46. Minati L, Torrengo S, Maniglio D, Migliaresi C, Speranza G. Luminescent graphene
quantum dots from oxidized multi-walled carbon nanotubes. Mater Chem Phys 2012;
137(1): 12–16.
47. Dong Y, Pang H, Ren S, Chen C, Chi Y, Yu T. Etching single-wall carbon nanotubes into
green and yellow single-layer graphene quantum dots. Carbon 2013; 64: 245–251.
48. Pan DY, Guo L, Zhang JC, Xi C, Xue Q, Huang H, Li JH, Zhang ZW, Yu WJ, Chen ZW,
Li Z, Wu MH. Cutting sp2 clusters in graphene sheets into colloidal graphene quantum
dots with strong green fluorescence. J Mater Chem 2012; 22(8): 3314–3318.
49. Tetsuka H, Asahi R, Nagoya A, Okamoto K, Tajima I, Ohta R, Okamoto A. Optically
tunable amino-functionalized graphene quantum dots. Adv Mater 2012; 24 (39),
5333–5338.
50. Zhu SJ, Zhang JH, Qiao CY, Tang SJ, Li YF, Yuan WJ, Li B, Tian L, Liu F, Hu R, Gao
HN, Wei HT, Zhang H, Sun HC, Yang B. Strongly green-photoluminescent graphene
quantum dots for bioimaging applications. Chem Commun 2011; 47(24): 6858–6860.
51. Zhou XJ, Zhang Y, Wang C, Wu XC, Yang YQ, Zheng B, Wu HX, Guo SW, Zhang JY.
Photo-Fenton reaction of graphene oxide: A new strategy to prepare graphene quantum
dots for DNA cleavage. ACS Nano 2012; 6(8): 6592–6599.
52. Dong Y, Shao J, Chen C, Li H, Wang R, Chi Y, Lin X, Chen G. Blue luminescent gra-
phene quantum dots and graphene oxide prepared by tuning the carbonization degree of
citric acid. Carbon 2012; 50(12): 4738–4743.

b2227_P2-V1_Ch-01.indd 17 23-Jun-17 2:54:11 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

18  J. Zhang & H. Lu

53. Qu D, Zheng M, Zhang L, Zhao H, Xie Z, Jing X, Haddad RE, Fan H, Sun Z. Formation
mechanism and optimization of highly luminescent N-doped graphene quantum dots. Sci
Rep 2014; 4: 5294.
54. Zhang B-X, Gao H, Li X-L. Synthesis and optical properties of nitrogen and sulfur
co-doped graphene quantum dots. New J Chem 2014; 38(9): 4615.
55. Tang L, Ji B, Li XM, Bai GX, Liu CP, Hao JH, Lin JY, Jiang HX, Teng KS, Yang ZB, Lau
SP. Deep ultraviolet to near-infrared emission and photoresponse in layered N‑doped
graphene quantum dots. ACS Nano 2014; 8(6): 6312–6320.
56. Liu R, Wu D, Feng X, Mullen K. Bottom-up fabrication of photoluminescent graphene
quantum dots with uniform morphology. J Am Chem Soc 2011; 133(39):
15221–15223.
57. Chen G, Zhuo Z, Ni K, Kim NY, Zhao Y, Chen Z, Xiang B, Yang L, Zhang Q, Lee Z, et
al. Rupturing C60 molecules into graphene-oxide-like quantum dots: Structure, photo-
luminescence, and catalytic application. Small 2015; 11(39): 5296–5304.
58. Chua CK, Sofer Z, Simek P, Jankovsky O, Klimova K, Bakardjieva S, Kuckova SH,
Pumera M. Synthesis of strongly fluorescent graphene quantum dots by cage-opening
buckminsterfullerene. ACS Nano 2015; 9(3): 2548–2555.
59. Yan X, Cui X, Li LS. Synthesis of large, stable colloidal graphene quantum dots with tun-
able size. J Am Chem Soc 2010; 132(17): 5944–5945.
60. Gupta V, Chaudhary N, Srivastava R, Sharma GD, Bhardwaj R, Chand S. Luminscent
graphene quantum dots for organic photovoltaic devices. J Am Chem Soc 2011; 133(26):
9960–9963.
61. Li N, Than A, Wang X, Xu S, Sun L, Duan H, Xu C, Chen P. Ultrasensitive profiling of
metabolites using tyramine-functionalized graphene quantum dots. ACS Nano 2016;
10(3): 3622–3629.
62. Luo P, Ji Z, Li C, Shi G. Aryl-modified graphene quantum dots with enhanced photolu-
minescence and improved pH tolerance. Nanoscale 2013; 5(16): 7361–7367.
63. Novak TG, Kim J, Song SH, Jun GH, Kim H, Jeong MS, Jeon S. Fast P3HT exciton
dissociation and absorption enhancement of organic solar cells by PEG-functionalized
graphene quantum dots. Small 2016; 12(8): 994–999.
64. Sun H, Wu L, Gao N, Ren J, Qu X. Improvement of photoluminescence of graphene
quantum dots with a biocompatible photochemical reduction pathway and its bioimaging
application. ACS Appl Mater Interfaces 2013; 5(3): 1174–1179.
65. Yang HB, Dong YQ, Wang X, Khoo SY, Liu B. Cesium carbonate functionalized gra-
phene quantum dots as stable electron-selective layer for improvement of inverted poly-
mer solar cells. ACS Appl Mater Interfaces 2014; 6(2): 1092–1099.
66. Yang S, Sun J, He P, Deng X, Wang Z, Hu C, Ding G, Xie X. Selenium doped graphene
quantum dots as an ultrasensitive redox fluorescent switch. Chem Mater 2015; 27(6):
2004–2011.
67. Jin SH, Kim DH, Jun GH, Hong SH, Jeon S. Tuning the photoluminescence of graphene
quantum dots through the charge transfer effect of functional groups. ACS Nano 2013;
7(2): 1239–1245.
68. Sun H, Gao N, Wu L, Ren J, Wei W, Qu X. Highly photoluminescent amino-functional-
ized graphene quantum dots used for sensing copper ions. Chem Eur J 2013; 19(40):
13362–13368.
69. Yan X, Li BS, Li LS. Colloidal graphene quantum dots with well-defined structures.
Accounts Chem Res 2013; 46(10): 2254–2262.

b2227_P2-V1_Ch-01.indd 18 23-Jun-17 2:54:11 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Chapter 2

Synthesis and Amino-Functionalization

of the Graphene Quantum Dots

Limin Dong*,†,# and Kejia Wu*

*College of Materials Science and Engineering

Harbin University of Science and Technology, Harbin, P.R. China
†
Key Laboratory of Engineering Dielectrics and Its Application
Ministry of Education
Harbin University of Science and Technology
Harbin, P.R. China
#
[email protected]

N,N-dimethyl formamide (DMF) was used as the ammonia source, and amino-
functionalized graphene quantum dots (af-GQDs) were prepared by the microwave-
solvothermal method. The samples were characterized in terms of morphological,
structural, and optical properties which are evaluated by X-ray diffraction (XRD),
transmission electron microscopy (TEM), the photoluminescence (PL) spectrum
and Fourier transform infrared (FTIR) spectra correspondingly. The graphene
oxide (GO) goes through with microwave expansion treatment first, then followed
by solvothermal process. Amino-functionalization has significant influence on the
crystallization degree and the layer spacing of graphene quantum dots (GQDs), and
the size was less than 10 nm, which was closed to zero-dimension state. The af-GQDs
have strong absorption in the ultraviolet region (λ ≤ 276 nm), but it has no significant
characteristic absorption peak, which fits with the semiconductor nanomaterials
ultraviolet absorption spectrum. The fluorescence emission spectrum of GQDs
that synthesized by this method perform excitation-dependent PL behaviors. The
excitation-dependent PL behaviors may be associated with electronic transitions
between electronic surface state level and the lowest unoccupied molecular orbital
(LUMO) energy level.

19

b2227_P2-V1_Ch-02.indd 19 23-Jun-17 2:54:49 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

20  L. Dong & K. Wu

1. Introduction
Nowadays, the main methods for preparing graphene quantum dots (GQDs)
included top-down1,2 and bottom-up strategies.3 Their principles were con-
trolling the size, but in the choice of raw materials was slightly different. The
top-down approach referred to cut the graphene sheets (GSs) (the large size)
by physical or chemical ways into the GQDs (the small size). It consisted of
hydrothermal methods,4 electrochemical strategies5 and chemical exfoliation6
of carbon fibers. It had the advantages of relatively simple steps, higher yield,
and defect of not being able to precisely control the morphological structure
and size of the GQDs. The bottom-up approach referred that using small
molecules carbon sources as precursors through a series of chemical reactions
prepared GQDs. The method mainly involved ultrasonic methods,7 solution
chemistry methods8 and microwave preparation.9 Most of the methods were
highly controllable, but were also complex and complicated. Moreover, the
strict preparation conditions of some special methods limited the promotion
of these methods. So, presently, the study of GQDs mainly was concentrated
on the improvement of preparation methods.
In recent years, the hydrothermal method in the preparation of nanom-
eter materials showed more obvious advantages. More and more researchers
are trying to adopt this method to prepare GQDs. But graphene as essential
material in these methods was complex, costly, and expensive. The yield of
GQDs is generally low with the graphene as the raw materials wasting
resources, so the choice of cheaper and easier materials to prepare GQDs is
currently a research focus in the field of synthetic process.
In the field of biomedical imaging, the luminous intensity of GQDs
directly affects the observation of labeled cells. Yang10 and his team found
that there are a lot of amino groups in the PEG graphene, which showed
good optical absorption and longer blood half-life in solution. According to
this conclusion, we considered bringing in amino in the GQDs, which obvi-
ously improves the PL intensity of the GQDs. It is beneficial to improve the
sensitivity of fluorescence analysis in biological studies and the observation of
labeled cells in the organism.
The fluorescence properties of the GQDs which take the amino-­
functionalization were significantly increased, and the level of cytotoxicity
was also the key factor to the af-GQDs application. Whether bringing in
amino enhances the combination of the QDs and non-specific cell or not, it
is a question worth considering. Chen11,12 used modified Photo-Fenton
hydrothermal method to prepare the fluorescence GQDs. He added

b2227_P2-V1_Ch-02.indd 20 23-Jun-17 2:54:49 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Synthesis and Amino-Functionalization of the Graphene Quantum Dots 21

a­ mmonium water in the process of preparation, and GQDs obtained actually

was af-GQDs. In this paper, the FT-IR analysis also proves this view. He
made a systematic study on the cytotoxicity of GQDs which he prepared.
Research showed that GQDs can be used as fluorescent probe and targeted
drug ­carrier. Meanwhile, test result showed that the toxicity of GQDs was
low, but it was not entirely non-poisonous.
Herein, we reported a facile microwave-assisted solvothermal approach to
the preparation of GQDs from GO nanosheets.13–15 We tested the fluorescence
properties of these GQDs, then prepared the af-GQDs.

2. Materials and Methods

In this chapter, we prepared GO via ultrasonic-assisted chemical oxidation, and
ultrasonic auxiliary operation started in the medium and high-temperature
reaction stages of modified Hummers method with secondary oxidation
­cutting of GO to obtain GO nanosheets. GQDs were prepared by microwave-
hydrothermal method. N,N-dimethyl formamide was used as the ammonia
source, and af-GQDs were prepared by the microwave-solvothermal method.

2.1. The preparation of GO

In this chapter, the preparation of GO was divided into three stages. Oxidant
potassium permanganate and acid (concentrated sulfuric acid and nitric acid)
was added in low-temperature reaction stage, oxide intercalated between the
layers, and temperature was controlled to 0–5°C. The second reaction stage
started with ultrasonic, and the reaction was warmed to 35–38°C, at which
deep oxidation happened, and the graphite layers exfoliation were sped up
under the action of ultrasonic. Ultrasonic continued in high temperature,
raising the reaction temperature to 82–85°C, at which deionized water was
added slowly for hydrolysis reaction of interlayer compounds. In order to
research the effect of ultrasonic in the process of GO preparation, modified
Hummers method was conducted as a contrast test.

2.2. The preparation of GQDs

The GQDs were prepared by microwave-hydrothermal method by changing
the experiment conditions, such as pH, hydrothermal temperature, hydro-
thermal time, and controlled the morphology, size, even fluorescent perfor-
mance of the product. The specific steps were as follows. Dissolve the drying

b2227_P2-V1_Ch-02.indd 21 23-Jun-17 2:54:49 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

22  L. Dong & K. Wu

GO nanosheets in water, ultrasonic stirring until it dissolved, and add sodium

hydroxide to adjust pH to different values to obtain mixed solution. The
solution was put into the microwave, regulating different heating power and
different times to obtain the reaction product. Then put the product into
polytetrafluoroethylene (PTFE) lined autoclave and heat in different tem-
peratures for a certain amount of time to obtain the next product. After the
reaction was completed, the supernatant was transferred into 1000 Da dialysis
tubing, and was dialyzed by deionized water for three times, then the GQDs
were obtained.

2.3. The preparation of af-GQDs

The GO nanosheets were synthesized by traditional Hummers method. It
was heated by a conventional microwave oven at a certain power (300, 500,
800, and 1000 W) for a period of time (1, 3, 5, 7, and 9 min) to complete
the pre-processing of precursor. After cooling to room temperature, the sam-
ple was dissolved by DMF. After doing ultrasonic treatment for several hours,
the mixture was transferred into PTFE lined autoclave and was heated in dif-
ferent temperatures for a certain amount of time. After the reaction was
completed, the supernatant was transferred into 1000 Da dialysis tubing, and
was dialyzed by deionized water for many times, then the af-GQDs were
obtained. The experimental parameters have a distinct effect on the growth
of GQDs such as microwave power, heating time, source concentration, etc.

3. Result and Discussion

3.1. Structure and morphology analysis of GO
Figure 1 shows the XRD spectra of GO prepared by ultrasonic-assisted
chemical oxidation and chemical oxidation, respectively. The (001) crystal
plane diffraction peaks of two samples both appeared, and diffraction peak of
GO prepared by ultrasonic-assisted chemical oxidation inclined left. According
to the Prague equation 2d sin θ = λ, calculations predicted that the interlayer
spacing of the material is 7.3 Å, larger than 6.9 Å that of GO prepared by
chemical oxidation, indicating that ultrasonic contributes to the exfoliation of
graphite layers.
Figure 2 shows the TEM images of the GO before and after pretreat-
ment. Figure 2(A) shows the GO prepared by the traditional Hummers; we
can find the exfoliation degree of graphite sheet is not complete, and the
thickness of GO is large. Figure 2(B) shows the GO prepared by the ultra-
sonic-assisted Hummers. We can find that the prepared GO looks like

b2227_P2-V1_Ch-02.indd 22 23-Jun-17 2:54:49 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Synthesis and Amino-Functionalization of the Graphene Quantum Dots 23

(001)

(001)

10 20 30 40 50
2u (°)

Figure 1. XRD spectra of GO. a. chemical oxidation, b. ultrasonic-assisted chemical oxidation.

Reprinted with permission from Ref. [15]. Copyright (2016) Virtual Institute of Physics.

Figure 2. TEM images of GO before and after processing. Reprinted with permission from
Ref. [15]. Copyright (2016) Virtual Institute of Physics.

b2227_P2-V1_Ch-02.indd 23 23-Jun-17 2:54:51 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

24  L. Dong & K. Wu

transparent film, and part of the GO wrinkles, these results indicate that the
oxidation of the graphite sheet is more thorough. As shown in Figure 2(C),
the reduction of GO is obtained after microwave heat reduction. It can be
found that agglomeration occurs since the hydrophilic oxygen-containing
groups on the surface of the graphene were removed. The stretch degree of
graphene is weaker than GO. Figure 2(D) shows the TEM spectrum of the
precursor after secondary oxidation process, its size is far smaller than the GO
before secondary oxidation process.
From Figure 2, we can see that with the improvement of the experimental
method, the layer thickness of GO changed significantly. Graphite was oxi-
dized and exfoliated more thoroughly after ultrasonic-assisted Hummers, and
the thinner GO is obtained. The external energy was added in the graphite
oxidation reaction stage, and the ultraphonic provides simultaneous, homoge-
neous, and fast heating. With the intercalation reactions of oxidants, the vibra-
tion frequency of sulfate and metal ions is increased, thus making them easier
to carry out intercalation between the graphite layers. The intercalation agents
were hydrolyzed to oxygen-containing group such as –COOH or –CHO
­during the latter part of the high-temperature hydrolysis.
The reduction of graphene oxide (RGO) is obtained after microwave heat
reduction. Compared with the GO, the RGO is nearly transparent, it means
the thickness is further decreased. This is because the microwave treatment
provided enough energy to the GO in a short time, and the unstable oxygen-
containing groups between the layers as well as at the edge of the graphite
were removed in the form of gases. After the secondary oxide, the size of the
GO decreased significantly. As shown in Figure 2(D), samples are in the form
of fragmented distribution, size of the GO is less than 100 nm. The reaction
process is in line with unzipping mechanism.16 In the process of acid oxida-
tion, bridging oxygen atom (C–O–C) tends to be arranged in line, this is easy
to disconnect the C–O bond. Once the epoxy bond was opened, it is easy to
form a more stable C=O/COOH group at room temperature. These groups
form epoxy chain on graphene nanosheets17 (Figure 2). These linear defects
make the GO easily breakable.

3.2. The GQDs were prepared by microwave-hydrothermal method

3.2.1. Structure and morphology analysis
Figure 3 shows the XRD pattern of the GQDs prepared by different prepara-
tion technologies Curve a shows the XRD pattern of GQDs which were
prepared by the hydrothermal reaction at 190°C for 10 h; when the GO
sheets were under 800 W for 9 min, and then the hydrothermal reaction was

b2227_P2-V1_Ch-02.indd 24 23-Jun-17 2:54:51 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Synthesis and Amino-Functionalization of the Graphene Quantum Dots 25

1200 (002)

900
b
Intensity (a.u.)

(002)

600
a

300

10 20 30 40 50 60 70 80
2u (°)

Figure 3. XRD spectra of GQDs prepared by different methods. Reprinted with permission
from Ref. [15]. Copyright (2016) Virtual Institute of Physics.

carried out at 190°C for 6 h, then we get the XRD pattern of GQDs
(curve b). From Figure 3, we can find that the intensity of the GQDs (002)
crystal plane diffraction peak increased significantly after microwave-assisted
hydrothermal method, and the crystallinity is also increased, this is consistent
with the TEM. At the same time, the diffraction peak of (002) crystal face is
from 27° to 24° to the left, the interplanar spacing is increased to 0.37 nm.
Microwave provides simultaneous, homogeneous, and fast heating in a short
time. Since the polarization effect of the microwave reaction reduces the acti-
vation energy of the reactants, thereby the rate of reaction is increased.
TEM characterization of the above samples was obtained as shown in
Figure 4. Figure 4(A) shows the TEM pattern of GQDs which were prepared
by the hydrothermal reaction at 190°C for 10 h; when the GO sheets were
prepared by microwave-assisted hydrothermal method, 800 W for 9 min, and
then the hydrothermal reaction was carried out at 190°C for 6 h, then we get
the TEM pattern of GQDs as shown in Figure 4(B). From Figure 4(B), we
can find the size of the GQDs is between 7 and 24 nm with microwave-
assisted, and the average size is about 20 nm, samples approximate spherical
particles, and we can see the lattice fringes. Without microwave processing,
the size of the GQDs is about 24 nm, the lattice fringe is not clear, the
­crystallinity is not high. Microwave processing can improve the crystallinity of
the sample, and the smaller graphene nanosheets can be obtained, these facili-
tate the formation of the GQDs.

b2227_P2-V1_Ch-02.indd 25 23-Jun-17 2:54:52 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

26  L. Dong & K. Wu

Figure 4. TEM images of GQDs prepared by different methods: (A) hydrothermal method,
(B) microwave-assisted hydrothermal method. Reprinted with permission from Ref. [15].
Copyright (2016) Virtual Institute of Physics.

GO

1731 1384 1091

1625

3431
Intensity (a.u.)

GQDs

1324 1025
1448

4000 3500 3000 2500 2000 1500 1000 500

Wavelength (nm)

Figure 5. FT-IR spectrums of GO and GQDs. Reprinted with permission from Ref. [15].
Copyright (2016) Virtual Institute of Physics.

3.2.2. Functional group analysis

Figure 5 shows the FT-IR pictures of GO and GQDs. As can be seen from
Figure 6, GO has many characteristic absorption peaks, they are located at
3440 cm–1 (υ–OH water), 1731 cm–1 (υc = o aldehyde), 1625 cm–1 (υc = o ketone),
1384 cm–1 (υ–CH3), 1091 cm–1 (υC–O–C ethers). Compared with the FT-IR
spectrum of GQDs, the stretching vibration at 1091 cm–1 (C–O–C) disap-
peared, while the C–O–C (aryl ether) stretching vibration peak at 1025 cm–1
appears. Hydroxyl stretching vibration peak appears at 1324 cm–1, and the

b2227_P2-V1_Ch-02.indd 26 23-Jun-17 2:54:53 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Synthesis and Amino-Functionalization of the Graphene Quantum Dots 27

800

314 nm 430 nm

600

257 nm
Intensity (a.u.)

400

200

0
200 250 300 350 400 450 500 550 600

Wavelength (nm)

Figure 6. Normalized fluorescence spectrum and ultraviolet visible absorption spectrum of

GQDs prepared by microwave-hydrothermal method. Reprinted with permission from Ref.
[15]. Copyright (2016) Virtual Institute of Physics.

bending vibration peak of the –CH2 appears at 1448 cm–1, while the stretch-
ing vibration peak of aldehyde group at 1729 cm–1 disappeared.
From the figure, we can see the disappearance of the aldehyde group, we
take the sodium hydroxide adjust the pH and introduce the hydroxyl , the
reaction of hydroxyl and hydrogen ions of aldehyde group to produce water,
and the –CH2–CO– is formed. Furans epoxy groups in the carbon–oxygen
bonds were broken, and the bridging oxygen atoms were removed, which is
the key to cut the GO into small-sized GQDs. During the experiment,
hydroxyl functional group introduced by sodium hydroxide has changed the
functional groups types of GQDs. The deprotonation process has a great
influence on the fluorescence properties of the GQDs.

3.2.3. Analysis of fluorescence properties

Figure 6 shows the normalized fluorescence spectrum and ultraviolet–visible
(UV–vis) absorption spectrum. GQDs maximum excitation wavelength is
314 nm, corresponding emission wavelength is 430 nm, and the stokes shift
is 116 nm. We can get the CIE coordinates through the emission peak data,
GQDs release blue fluorescence by the chromaticity diagram analysis. GQDs
have two excitation peaks located at 257 and 314 nm, and its fluorescence
emission peaks are all located at 430 nm, the emission band is due to the

b2227_P2-V1_Ch-02.indd 27 23-Jun-17 2:54:54 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

28  L. Dong & K. Wu

Figure 7. Electron transition diagram of GQDs triplet state. Reprinted with permission from
Ref. [15]. Copyright (2016) Virtual Institute of Physics.

σ→π* and π→π* transition. UV absorption spectrum shows that GQDs have
absorption in the ultraviolet region, which is due to the σ→π* transition of
sp2 aromatic group.
The fluorescence properties of GQDs are related to the carbene structure
of triplet electron transition. For triplet σ1π1 structure, σ and π orbital were
occupied by unpaired electrons. In the process of triplet electron transition,
the electrons from the σ and π orbital track (highest occupied molecular
orbital, HOMO) jump into the π* orbital (lowest unoccupied molecular
orbital, LUMO). In GQD emission process, electrons from π orbital jump
into the LUMO energy levels, and the energy required is 4.15 eV (314 nm).
Electrons from the σ orbital jump into the LUMO energy levels, the energy
required is 5.07 eV (257 nm). As shown in Figure 7, the energy gap (δE)
between the σ and π is 0.92 eV, and the value is consistent with the triplet
carbene theoretical value δE (<1.5 eV).18 Therefore, when the energy reaches
the energy required for electron transitions from σ and π orbital to the
LUMO, electron transitions will occur; fluorescence is generated when the
energy changed from the LUMO to the lower energy state.

3.3. Analysis of af-GQDs

DMF was used as the ammonia source and solvent, af-GQDs were prepared
by the microwave-solvothermal method.

3.3.1. Structure and morphology analysis

Figure 8, shows the XRD pattern of the precursor, GQDs and af-GQDs.
(002) crystal face diffraction peak of the af-GQDs was located at 25.7°. The
diffraction peak of GQDs (002) crystal face is at 24°, the peak shape looks

b2227_P2-V1_Ch-02.indd 28 23-Jun-17 2:54:54 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Synthesis and Amino-Functionalization of the Graphene Quantum Dots 29

(001) (002)

(002)
af-GQDs
Intensity (a.u.)

GQDs

GO

10 20 30 40 50
2 θ (°)

Figure 8. XRD spectra of the precursor, GQDs and af-GQDs. Reprinted with permission
from Ref. [15]. Copyright (2016) Virtual Institute of Physics.

like bread peak, and the peak is not obvious. The diffraction peak of GO
(001) crystal face is located at about 10°, that’s a strong and sharp peak. The
figure shows that the intensity of the af-GQDs (002) crystal plane diffraction
peak is greater than GQDs, the corresponding 2θ diffraction angle is right
shift to 25.7°, and the interplanar spacing is reduced to 0.35 nm. Amino-
functionalization has significant influence on the crystallization degree and
the layer spacing of GQDs. We analyze that when the nitrogen source reacted
with oxygen-containing functional groups in the GS, and it easily reacted with
the functional groups on the edge. Unreacted oxygen-containing functional
groups and bridging oxygen atom are transformed into water or gas, etc., and
they are removed under the high-temperature and high-pressure atmosphere.
This makes the graphene defects decreased, the graphene crystal structure
tends to complete. Af-GQDs interplanar spacing (0.35 nm) is less than the
GO (002) interplanar spacing (0.431 nm) because the oxygen-­containing
groups between the graphite layers are removed during the ­reaction at high-
temperature and pressure.
Figure 9 shows the TEM images of af-GQDs. Figure 9(A) is an enlarged
10,000 times TEM image of the af-GQDs, and Figure 9(B) is an enlarged
60,000 times TEM image of the af-GQDs. The af-GQDs dispersed uniformly
without agglomeration. From the high-resolution electron microscope photo-
graph (HRTEM), we can see that the size of the prepared af-GQDs are about 5–7
nm, it is close to the zero-dimensional (0D) state. The (002) crystal face lattice
fringes of the af-GQDs are legible, and the interplanar spacing is about 0.35 nm.

b2227_P2-V1_Ch-02.indd 29 23-Jun-17 2:54:55 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

30  L. Dong & K. Wu

Figure 9. TEM images of af-GQDs. Reprinted with permission from Ref. [15]. Copyright
(2016) Virtual Institute of Physics.

benzene
GQDs nuclear
Intensity (a.u.)

–OH
af-GQDs C=O
R–NH
Ammonium ion
Absorption band
CH2 C–N
NH
R-N NH
NH2 (wagging
–OH R=O CH3 vibration)

3500 3000 2500 2000 1500 1000 500

Wavelength (nm)

Figure 10. FT-IR spectrums of GQDs and af-GQDs. Reprinted with permission from Ref.
[15]. Copyright (2016) Virtual Institute of Physics.

3.3.2. Functional group analysis

Figure 10, shows the FT-IR spectrums of GQDs and af-GQDs. After amino-
functional modification, the types of af-GQDs functional groups increased
significantly. The stretching vibration of CH2 appears at 2925 cm–1, the
absorption band of the sp2 hybridized carbon ring (benzene nucleus for
short) appears at 1448 cm–1. It means that the GQDs structural defects
decreased after microwave-assisted hydrothermal, and crystal structure tends
to complete. In addition, the hydroxyl weakened and disappeared with
microwave-solvothermal reaction, the number of oxygen-containing groups

b2227_P2-V1_Ch-02.indd 30 23-Jun-17 2:54:56 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Synthesis and Amino-Functionalization of the Graphene Quantum Dots 31

is also reduced. After a series of reaction, the NH2 and C–N bending or
stretching vibration peaks appear at 2362, 1492, 1263, and 698 cm–1. It
means that nitrogen-containing functional groups were introduced.

3.3.3. Fluorescence Performance Analysis

Af-GQDs have a wealth of fluorescence emission performance. Figure 11,
shows the UV–vis absorption and fluorescence spectra of af-GQDs. The af-
GQDs have strong absorption in the ultraviolet region (λ ≤ 276 nm), but it
has no significant characteristic absorption peak, which fits with the semicon-
ductor nanomaterials ultraviolet absorption spectrum. In addition, a shoulder
peak at 276 nm indicates the existence of functional groups with lone pair
electrons (such as carboxyl, etc.). In addition, af-GQDs absorbance decreased
at 494 nm, while the maximum emission wavelength is located at 494 nm.
After amino-functional modification, the PL spectra of af-GQDs changed
significantly. We find the maximum excitation and emission peak red-shift at
494 and 416 nm respectively (416 nm excitation), and the stokes shift is
78 nm. The emission wavelength data was imported with CIE color coordi-
nates and calculated, its color coordinate is located in green light-emitting
region. Emission wavelength red-shift indicates the energy of the electronic
transitions is decreased. After amino-functional modification, the electronic

416 nm 494 nm

PLE
383 nm
Abs
337 nm PL
Intensity (a.u.)

281 nm

250 300 350 400 450 500 550 600 650

Wavelength (nm)

Figure 11. UV–vis absorption and fluorescence spectra of af-GQDs. Reprinted with permis-
sion from Ref. [15]. Copyright (2016) Virtual Institute of Physics.

b2227_P2-V1_Ch-02.indd 31 23-Jun-17 2:54:57 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

32  L. Dong & K. Wu

1000

600 420 nm
800
440 nm
500
460 nm

Intensity (a.u.)
Intensity (a.u.)

400 416 nm 600

383 nm 480 nm
300 337 nm
400 500 nm
281 nm
200
200
100

0 0
400 450 500 550 600 650 450 500 550 600 650 700
Wavelength (nm) Wavelength (nm)
(A) (B)

Figure 12. The fluorescence emission spectra of the af-GQDs excited by different excitation
wavelengths. Reprinted with permission from Ref. [15]. Copyright (2016) Virtual Institute of
Physics.

structure of GQDs changed, the bandgap between the HOMO and the
LUMO is shortened. We analyze that amino groups were introduced into the
GQDs edge. While reacting with amino group electron orbit, graphene mol-
ecule delocalized π electrons HOMO orbital is moved to the higher energy
orbits, and the bandgap is shortened consequently.
Af-GQDs excitation spectra have four excitation peaks, and they are
located at 281, 337, 383, and 416 nm, respectively. Figure 12(A) shows the
emission spectra of af-GQDs excited by different excitation wavelengths.
Although the excitation wavelength changes, the corresponding af-GQDs
emission peak wavelength does not change. The maximum emission peak
wavelength is about 490 nm. The research shows that with the increase of the
intensity of each excitation peak, the intensities of emission peaks are also
increased. When excited by the 416 nm, the highest emission peak intensity
is obtained. GQDs have an emission characteristic of multiwavelength excita-
tion, we analyze that apart from the σ → π * and π → π * electronic transition
of GQDs carbene structure itself, since the introduction of the nitrogen-
containing functional groups, the electron distribution of the af-GQDs sur-
face is changed.
To study the relationship between the emission spectrum and excitation
wavelength, the af-GQDs were tested by the different excitation wavelengths.
Figure 12(B) shows the emission spectra of af-GQDs which were excitated
by different wavelengths. The inset at the right shows the af-GQDs emitting

b2227_P2-V1_Ch-02.indd 32 23-Jun-17 2:54:58 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Synthesis and Amino-Functionalization of the Graphene Quantum Dots 33

light colors which were excitated by the 420, 440, 460, 480, and 500 nm,
respectively. With the excitation wavelength changed from 420 to 500 nm,
the GQDs emission peak wavelength from 497 nm red-shift to 546 nm, and
the emission intensity is followed by decline. The fluorescence emission
­spectrum of GQDs that synthesized by this method perform excitation-
dependent PL behaviors. The excitation-dependent PL behaviors may be
associated with electronic transitions between electronic surface state level
and the LUMO energy level.

4. Influence of Experimental Conditions of GQDs

4.1. Effect of the microwave power
Figure 13, shows the XRD patterns of RGO and GQDs under different
microwave power in the microwave. We can learn that with the increase of
microwave power, the (002) diffraction peak of GQDs is close to the (002)
diffraction peak of graphene, and the crystal structure of the GQDs is close
to the crystal structure of the graphene too. When the power is up to 800 W,
the crystal structure of GQDs is close to the crystal structure of graphene.
When the power continues to increase, (002) diffraction peak appears left
shift. The reason for this phenomenon may be that the GO was further
­carbonized because of the excessive microwave power.

RGO

300 W

500 W

800 W

1000 W

20 40 60 80
2u (°)

Figure 13. XRD spectra of GQDs which conducted at different microwave power. Reprinted
with permission from Ref. [14]. Copyright (2016) Virtual Institute of Physics.

b2227_P2-V1_Ch-02.indd 33 23-Jun-17 2:54:58 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

34  L. Dong & K. Wu

450
483 nm
400

350 485 nm
300 500 w
Intensity (a.u.)

457 nm
250 466 nm 300 w
200 438 nm 0

150 800 w
100 1000 w

50

0
400 450 500 550 600 650
Wavelength (nm)

Figure 14. The fluorescence emission spectra of GQDs under different microwave power.
Reprinted with permission from Ref. [14]. Copyright (2016) Virtual Institute of Physics.

Microwave power has a significant effect on the fluorescence properties of

GQDs (Figure 14). The microwave time is set to 3 min, and the GO sheets are
carried out under different microwave power. With the increase of the micro-
wave power, the fluorescence intensity of GQDs first increased and then
decreased, when the microwave power was set to 500 W, the intensity got the
strongest. With the increase of microwave power, interlamellar spacing of GO is
constantly increasing. In the process of reaction, bridging oxygen atoms have
been removed, which promoted the fracture of the oxygen-containing groups.
We can get smaller size QDs, it can also improve the quantum yield, fluorescence
intensity then increased. But when the microwave power continues to increase,
a portion of GO was carbonized, and the fluorescence intensity decreased.

4.2. Effect of the microwave time

Figure 15, shows the XRD patterns of RGO and GQDs that are under dif-
ferent microwave times. We can see that (001) crystal plane diffraction peak
of GQDs is about 7.8°. According to Bragg formula (1), GQDs interlamellar
spacing: d = 11.33 Å. (002) crystal surface appears at approximately 24°, it
was the characteristic diffraction peaks of graphene. With the increase of
microwave time, the diffraction peak of GQDs is close to the standard diffrac-
tion peak of graphene (26°), and the crystal structure of the GQDs is close
to the crystal structure of the graphene as well. The microwave power is set
to 800 W power here.
Microwave time also has a significant effect on the fluorescence properties
of GQDs. GO sheets are under microwave power of 500 W for different

b2227_P2-V1_Ch-02.indd 34 23-Jun-17 2:54:59 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Synthesis and Amino-Functionalization of the Graphene Quantum Dots 35

Intensity (a.u.)
RGO

3 min
5 min

7 min
9 min

10 20 30 40 50 60 70 80
2u (°)

Figure 15. XRD spectra of RGO and GQDs under different microwave times. Reprinted
with permission from Ref. [14]. Copyright (2016) Virtual Institute of Physics.

5 min

3 min
300
0
Intensity (a.u.)

7 min
200

9 min
100

0
350 400 450 500 550 600 650
Wavelength (nm)

Figure 16. The fluorescence emission spectra of GQDs under different microwave times.
Reprinted with permission from Ref. [14]. Copyright (2016) Virtual Institute of Physics.

times. Figure 16, shows the emission spectrum of samples. With the increase
in the microwave time, the fluorescence intensity of GQDs first increased and
then decreased; when the microwave time was set to 5 min, the intensity got
the strongest. The appropriate microwave power and time can improve the
fluorescence properties of GQDs. Besides, it can also improve the quantum
yield, so the fluorescence intensity of GQDs increased. But the high concen-
tration of GQDs in the solution leads to concentration quenching effect, and
the fluorescence intensity decreased instead.

b2227_P2-V1_Ch-02.indd 35 23-Jun-17 2:55:01 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

36  L. Dong & K. Wu

450
483 nm
400

350 480 nm 170˚C

300 482 nm 180˚C
Intensity (a.u.)

481 nm
250 190˚C
160˚C
200

150

100

50

0
400 450 500 550 600 650
Wavelength (nm)

Figure 17. The fluorescence emission spectra of GQDs at different solvothermal tempera-
tures. Reprinted with permission from Ref. [14]. Copyright (2016) Virtual Institute of Physics.

4.3. The effect of the solvothermal temperature

Figure 17, shows the fluorescence emission spectra of GQDs at the different
solvothermal temperatures (160, 170, 180, and 190°C). Temperature has no
effect on the fluorescence emission wavelength of the GQDs. With the
increase in the solvothermal temperature, the fluorescence intensity of GQDs
first increased and then decreased. The yield of GQDs is on the increase when
the temperature increases. But when the temperature continues to increase, a
portion of GO was carbonized, and then the fluorescence intensity decreased.

5. Conclusion
This paper focuses on the preparation and fluorescence properties of GQDs,
and the preparation of the af-GQDs to enrich fluorescence properties. Finally,
the paper reaches the following conclusions:

(1) Highly luminescence GQDs were prepared by the microwave-assisted

hydrothermal method. The operation process was simplified, and the
time of hydrothermal reaction was shortened from 10 to 6 h. The aver-
age size of prepared GQDs was 20 nm. The (002) crystal face lattice
fringes of the GQDs was legible, and the interplanar spacing was about
0.37 nm. FT-IR figure showed that GQDs contains hydroxyl, carboxyl,
and bridging oxygen atoms. PL spectrum indicated the maximum

b2227_P2-V1_Ch-02.indd 36 23-Jun-17 2:55:02 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Synthesis and Amino-Functionalization of the Graphene Quantum Dots 37

excitation wavelength was 314 nm and the emission peak wavelength

was 430 nm.
(2) DMF was used as the ammonia source and solvent, and af-GQDs
were prepared by the microwave-solvothermal method. The af-GQDs
­dispersed uniformly without agglomeration. From the HRTEM, we can
see that the size of the prepared af-GQDs were about 6 nm, and it was
close to the 0D state. The interplanar spacing of (002) crystal face was
about 0.35 nm. From the FT-IR figure, the NH2 and C–N bending or
stretching vibration peaks could be found. The maximum emission peak
was 494 nm (416 nm excitation), and the stokes shift was 78 nm. The
change of emission and excitation was caused by delocalized π orbital
and nitrogen-containing functional groups. We also verified the excita-
tion wavelength dependence of af-GQDs.
(3) Based on fluorescence performances and TEM pictures, it was concluded
that ideal reaction conditions were as follows: microwave power was
800 W, and microwave processing time 10 min, and hydrothermal time
6 h, and hydrothermal temperature 170°C.

References
1. Li Y, Zhao Y, Cheng H, et al. Nitrogen-doped graphene quantum dots with oxygen-rich
functional groups. J Am Chem Soc 2011; 134(1): 15–18.
2. Shen J, Zhu Y, Chen C, et al. Facile preparation and upconversion luminescence of
­graphene quantum dots. Chem Commun 2011; 47(9): 2580–2582.
3. Liu R, Wu D, Feng X, et al. Bottom-up fabrication of photoluminescent graphene
quantum dots with uniform morphology. J Am Chem Soc 2011; 133(39):
­
15221–15223.
4. Lu J, Yeo PSE, Gan CK, et al. Transforming C60 molecules into graphene quantum dots.
Nat Nanotechnol 2011; 6(4): 247–252.
5. Li Y, Hu Y, Zhao Y, et al. An electrochemical avenue to green-luminescent graphene
quantum dots as potential electron-acceptors for photovoltaics. Adv Mater 2011; 23(6):
776–780.
6. Schnez S, Molitor F, Stampfer C, et al. Observation of excited states in a graphene
­quantum dot. Appl Phys Lett 2009; 94(1): 012107.
7. Zhuo S, Shao M, Lee ST. Upconversion and downconversion fluorescent graphene quan-
tum dots: ultrasonic preparation and photocatalysis. Acs Nano 2012; 6(2): 1059–1064.
8. Yan X, Cui X, Li B, et al. Large, solution-processable graphene quantum dots as light
absorbers for photovoltaics. Nano Lett 2010; 10(5): 1869–1873.
9. Li LL, Ji J, Fei R, et al. A facile microwave avenue to electrochemiluminescent two-color
graphene quantum dots. Adv Funct Mater 2012; 22(14): 2971–2979.
10. Yang K, Zhang S, Zhang G, et al. Graphene in mice: ultrahigh in vivo tumor uptake and
efficient photothermal therapy. Nano Lett 2010; 10(9): 3318–3323.

b2227_P2-V1_Ch-02.indd 37 23-Jun-17 2:55:02 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

38  L. Dong & K. Wu

11. Chen Y, Qi Y, Liu B. Polyacrylic acid functionalized nanographene as a nanocarrier for

loading and controlled release of doxorubicin hydrochloride. J Nanomater 2013;
2013(5): 3805–3816.
12. Chen Y, Qi Y, Yan X, et al. Green fabrication of porous chitosan/graphene oxide
­composite xerogels for drug delivery. J Appl Polym Sci 2014; 131(6): 596–602.
13. Cui YQ, Wu Z, Dong LM, et al. Graphene oxide prepared via ultrasonic assisted chemical
oxidation method and its fluorescence property. Adv Mater Res 2014; 986: 84–87.
14. Dong LM, Shi DY, Wu Z, et al. Improved solvothermal method for cutting graphene
oxide into graphene quantum dots. Dig J Nanomater Bios 2015; 10(3): 855–864.
15. Dong LM, Wu KJ, Shi DY, et al. Graphene quantum dots with its amino-functionalization
prepared by microwave-hydrothermal Method. Dig J Nanomater Bios 2015; 10(4):
1215–1227.
16. Kosynkin DV, Higginbotham AL, Sinitskii A, et al. Longitudinal unzipping of carbon
nanotubes to form graphene nanoribbons. Nature 2009; 458(7240): 872–876.
17. Li Z, Zhang W, Luo Y, et al. How graphene is cut upon oxidation? J Am Chem Soc 2009;
131(18): 6320–6321.
18. Bourissou D, Guerret O, Gabbai FP, et al. Stable carbenes. Chem Rev 2000; 100(1):
39–92.

b2227_P2-V1_Ch-02.indd 38 23-Jun-17 2:55:02 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Chapter 3

Plasmons in Graphene Quantum Dots

Haifeng Yin

College of Physics and Electronic Engineering, Kaili University

Kaili, Guizhou 556011, P.R. China
[email protected]

Compared with plasmons in metals, plasmons in graphene have more advantages.

Thereinto, the carrier density of graphene can be tuned via electrostatic gating or
chemical doping, which provides the highly desired feature of tunability to plasmonic
materials. These advantages make graphene a very promising plasmonic material that
can complement existing plasmonic materials in a broad electromagnetic spectrum
range. In this chapter, we start with a general description of the plasmon behavior
of the homogeneous graphene. Then, we investigated plasmons in pristine graphene
quantum dots (GQDs). Compared with the homogeneous graphene, in the lower-
energy resonance zone, spectral band is greatly broadening, and the photoabsorption
strength line splits due to the effect of the size and the confinement. We further
discussed plasmons in doped GQDs, and plasmon excitations in GQD dimers.

1. Introduction of Graphene Plasmon

Graphene, a two-dimensional (2D) form of carbon in which the atoms are
arranged in a honeycomb lattice,1 has already been shown to possess unique
electric, mechanical, magnetic, and thermal properties with a multitude of
exciting applications that are being vigorously pursued by industry and aca-
demia. More interestingly, it is in optics where graphene has shown its “true
colors”. Graphene has an extremely high-quantum efficiency for light–matter
interactions, and contains plasmons with unusual properties.

39

b2227_P2-V1_Ch-03.indd 39 23-Jun-17 2:55:27 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

40  H. Yin

Graphene surface plasmons provide an appealing alternative to noble-

metal plasmons because they display a range of potentially useful properties.
Graphene surface plasmons exhibit a wavelength compression factor of the
plasmon wavelength with respect to the free-space wavelength ∼200–300,2
one to two orders of magnitude higher than for noble metals.2 Graphene
surface plasmon exhibit relatively long propagation distances as compared to
surface plasmon modes in noble metals. The frequencies of surface plasmon
polariton modes are highly tunable via electrostatic gating.3,4 This property
can be used to direct plasmons along switchable paths controlled by electro-
static gates, representing a unique capability not present in metals.
Furthermore, the propagation of graphene plasmons was reported by Refs. 3
and 4, where real-space visualization of mid-infrared graphene plasmons was
revealed.4 By employing scattering-type near-field microscopy, it was possible
to excite and spatially image propagating and localized plasmons in tapered
graphene nanoribbon at infrared frequencies.3,4
Graphene can be combined with plasmonic metamaterials.5 The light
interaction is modulated and enhanced by placing arrays of metal particles or
antennas on the graphene surface. The combination of graphene photonics
with plasmonics can improve the performance of existing devices, overcom-
ing some limitations associated with the transparency of graphene in the vis-
ible near-infrared (NIR). GQDs which are sensitized by semiconductor
quantum dots (QDs) can lead to strong photovoltage enhancements.6 By
modulation of the metal dielectric function, graphene-plasmonics could pave
the way towards novel sensing routes.

2. Plasmons in GQDs
2.1. Plasmons in pristine GQDs
Collective electron oscillations in QDs form surface plasmon resonances,7–9
which exhibit unusual properties in energy, lifetime, optical spectroscopy, and
short-time dynamics. Surface plasmon can be excited by the electromagnetic
field of light. In the case of metallic nanoparticles, where the electrons are
confined in the three dimensions, the electron oscillations induce an electric
field around the nanoparticles that can be much larger than the incident light
one. Surface plasmon resonances are one of the best examples that things are
different at the nanoscale. When the size of a particle is reduced to a few
nanometres, the optical properties are dramatically modified by the appear-
ance of surface plasmons and its resulting behavior is completely different
from the bulk one. Surface plasmons open the possibility to amplify, concen-
trate and manipulate light at the nanoscale, overcoming the diffraction limit

b2227_P2-V1_Ch-03.indd 40 23-Jun-17 2:55:27 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Plasmons in Graphene Quantum Dots 41

of traditional optics and increasing resolution and sensitivity of optical

probes.10,11 Consequently, surface plasmons can be used in a wide range of
fields, including biomedical,12 energy,13 environment protection,14 sensing
and information technology applications.15 Nowadays, there are well-estab-
lished applications of surface plasmons that increase rapidly with the develop-
ment of our capabilities to fabricate and manipulate nanomaterials.
Recently, plasmon excitations in GQDs have attracted much attention in
nanosciences.16–18 Wang et al. investigated graphene edge plasmons in finite
GQDs.16 They considered a low electron density edge layer and approximated
it by a linear profile. Based on the semiclassical conductivity, they found that
graphene edge plasmons behave analogously to those in 2D electron gas for
abrupt edges, but important distinctions arise for gradual edge profiles. In
particular, graphene supports fewer edge modes than a 2D electron gas at a
given wave vector q. Mishchenko and co-workers investigated spectra of plas-
mons in spatially inhomogeneous graphene flakes by the hydrodynamic
approach.17 They also found that plasmon waves are confined to the bound-
ary region due to spatial separation of electrons and holes in graphene, and
demonstrated that guided plasmon modes can be easily controlled by the
external electric field. It is well known that the edge structure of GQDs (zig-
zag- or armchair-edge configurations) greatly influences their electro-optical
properties. In these studies, the influence of different atomic structures of
graphene edges is not considered. The research of the influence of different
edge states is also helpful to understand the mechanism of plasmon excita-
tions in GQDs.
Using time-dependent density functional theory, Yin et al. investigated
the collectivity of the electronic motion in GQDs.19,20 There are still two main
surface plasmon bands in these GQDs, one around 16eV (π + σ plasmon) and
the other around 5 eV (π plasmon), as shown in Figure 1. Compared with the
homogeneous graphene, in the lower-energy resonance zone, spectral band is
greatly broadening, even extending to the NIR spectroscopy range (around
1 eV), and the photoabsorption strength line splits due to the effect of the
size and the confinement. There are strong plasmon resonances in the visible
region of the spectrum of GQDs. The edge configuration plays an important
role in the absorption spectrum of GQDs. When the impulse excitation polar-
ized in the armchair-edge direction, there is a relatively larger range of
absorption band in the lower-energy resonance zone, and the width (along
the Y-axis direction) of QDs also has an influence on absorption spectrum. In
the low-energy resonance zone, the spectrum is red-shifted with the increase
of nanostructure length in the absorption direction. This phenomenon shows
that the π plasmon resonance mode is a long-range charge transfer excitation.
The induced charge of most low-energy resonance points is distributed in the

b2227_P2-V1_Ch-03.indd 41 23-Jun-17 2:55:27 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

42  H. Yin

Figure 1. The dipole response (optical) of the rectangular GQDs to an impulse excitation
polarized in the X-axis (A) and Y-axis (B) direction. The inset is the schematic diagram of the
GQDs. Fourier transform of the induced charge density for GQD (A) and (B) at the energy
resonance points 5.13 and 3.37 eV, respectively, as shown in the direction of the arrow.20

boundary region. Moreover, in the hexagonal GQDs, there is a reverse two-

dipole excitation mode. The formation of the reverse two-dipole excitation
mode is mainly due to the inducement of the external electric field and the
shielding effect.
Using the time-dependent density functional theory, Yin et al. also inves-
tigated the plasmon excitation in ring GQDs, as shown in Figure 2.21 In
lower-energy resonance region, compared with the same size GQD, the
main absorption peak of spectra of ring GQDs shows red-shift. There are
two main plasmon resonance modes in ring GQDs, which are respectively
low-energy bonding mode and high-energy anti-bonding mode. Moreover,
to a certain extent, the plasmon excitation in ring GQDs depends on the size
of the system.
The pathways of plasmon losing its energy (or damping) play a pivotal
role in plasmonic technology and science. Yan et al. studied damping path-
ways of mid-infrared plasmons in GQDs, and revealed damping channels via
graphene intrinsic optical phonons and scattering from the edges.22 Plasmon
lifetimes of 20 fs or less are observed when damping via the emission of gra-
phene optical phonons is allowed. Surface polar phonons in the SiO2 sub-
strate under graphene nanostructures lead to a significantly modified

b2227_P2-V1_Ch-03.indd 42 23-Jun-17 2:55:29 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Plasmons in Graphene Quantum Dots 43

Figure 2. Optical absorption of different hexagonal ring GQDs to an impulse excitation

­polarized in the X-axis direction. The insets are schematic diagrams of these QDs.21

plasmon dispersion and damping in contrast to the case of a non-polar

diamond-like carbon substrate. The study paves the way for applications of
graphene in plasmonic modulators, waveguides and detectors from sub-
terahertz to mid-infrared regimes. Moreover, when driven by light, the opti-
cal response is dictated by the dielectric environment and geometrical param-
eters. Therefore, plasmons are extremely important for sensing applications.
Plasmons in graphene disks have the additional benefit of being highly tun-
able via electrical stimulation. Mechanical vibrations create structural defor-
mations in ways where the excitation of localized surface plasmons can be
strongly modulated. The study of Wang et al. shows that the spectral shift in
such a scenario is determined by a complex interplay between the symmetry
and shape of the modal vibrations and the plasmonic mode pattern.23 Tuning
confined modes of light in graphene via acoustic excitations paves new ave-
nues in shaping the sensitivity of plasmonic detectors, and in the enhance-
ment of the interaction with optical emitters, such as molecules, for future
nanophotonic devices.

b2227_P2-V1_Ch-03.indd 43 23-Jun-17 2:55:29 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

44  H. Yin

2.2. Plasmons in doped GQDs

Plasmons in doped graphene have more advantages due to the carrier density
of graphene can being tuned, which make graphene a very promising
­plasmonic material that can complement existing plasmonic materials in a
broad electromagnetic spectrum range.24,25 Electrically doped graphene has
been found to support long-lived plasmonic excitations that efficiently
­couple to light and are actively tunable by changing the density of charge
carriers. The study of Manjavacas et al. shows that chemically synthesized
polycyclic aromatic hydrocarbons exhibit molecular plasmon resonances that
are remarkably sensitive to the net charge state of the molecule and the
atomic structure of the edges.26 These molecules can be regarded as nanome-
ter-sized forms of graphene, from which they inherit their high-electrical
tunability. The addition or removal of a single-electron can switche these
molecular plasmons on/off. They also studied the interaction between plas-
mons in neighboring molecules. Their findings suggest new paradigms for
electro-optical modulation and switching, single-electron detection, and
sensing using individual molecules.
Plasmon-assisted second-harmonic generation and down conversion with
good efficiencies at the few-photon level have been argued to be possible
when the fundamental and the second-harmonic are simultaneously resonant
with plasmons in a nanographene.27 Cox et al. have predicted that a similar
mechanism can lead to unprecedentedly intense second-harmonic generation
and third-harmonic generation.28 Cox et al. also found that doped graphene
nanoislands present unique opportunities for enhancing nonlinear optical
wave-mixing processes between two externally applied opticalfields at the
nanoscale.25 These small islands can support pronounced plasmons at multi-
ple frequencies, resulting in extraordinarily high wave-mixing susceptibilities
when one or more of the input or output frequencies coincide with a plasmon
resonance. By varying the doping charge density in a nanoisland with a fixed
geometry, enhanced wave mixing can be realized over a wide spectral range
in the visible and NIR. Their calculations for armchair-graphene triangles
composed of up to several hundred carbon atoms display large wave mixing
polarizabilities compared with metal nanoparticles of similar lateral size, thus
supporting nanographene as an excellent material for tunable nonlinear opti-
cal nanodevices.
Though electrostatic gating is one well-established method, chemical
doping can potentially achieve the required control without the use of exter-
nal voltages. Therefore, in chemical-doped graphene, tunable plasmons have

b2227_P2-V1_Ch-03.indd 44 23-Jun-17 2:55:29 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Plasmons in Graphene Quantum Dots 45

been receiving more and more attention.29 Nitrogen is considered to be an

excellent element for the chemical doping of graphene because it has compa-
rable atomic size and contains five valence electrons for bonding with carbon
atoms.30,31 In monolayer graphene, experimentally, different methods have
been successfully used to control the nitrogen doping concentration and con-
figuration.32–35 Theoretically, the stable configuration and the unique elec-
tronic properties of nitrogen-doped graphene have also been studied.36 These
studies indicate that nitrogen-doped graphene is the promising optical and
electronic device.36
Recently, people pay more and more attention to the nitrogen-doped
GQDs due to its marvelous properties associated with quantum-confinement
and edge effects.37 Experimentally, significant advancement has been made in
the synthesis of nitrogen-doped GQDs.38–41 For example, Li et al. developed
a hydrothermal approach for the synthesis of nitrogen-doped GQDs by cut-
ting N-doped graphene.34 They also investigated optical properties of the
nitrogen-doped GQDs and found, in the nitrogen-doped GQDs, the photo-
luminescence (PL) blue-shift. Jin et al. successfully fabricated another kind of
nitrogen-doped GQDs which are functionalized with amine groups.41
Using the time-dependent density functional theory, Yin et al. have car-
ried out a systematic study of collective excitations of nitrogen-doped GQDs,
and mainly investigated the impacts of the pyridinic and substitutional nitro-
gen doping on the plasmon excitations.42 First of all, the pyridinic nitrogen
doping does not affect the plasmon excitation characteristic of graphene
nanostructures, as shown in Figure 3. Absorption spectra of the pyridine-like
nitrogen-doped NGQD and the pristine GQD are almost identical. Second,
for substitutional nitrogen doping, the low-energy plasmon resonances of
GQDs mainly depend on the competition between the reduced symmetry of
the QDs which is brought by doping nitrogen and the relative increase of the
electron densities as shown in Figures 4 and 5. Compared with the absorption
spectrum of the pristine hexagonal GQD, low-energy absorption spectra of
substitutional nitrogen-doped hexagonal GQDs are red-shifted, which is
mainly due to the reduced symmetry of the QDs. Unlike substitutional
nitrogen-doped hexagonal GQDs, along the armchair-edge direction, the
main lower-energy absorption peaks of substitutional nitrogen-doped rectan-
gular GQDs are always blue-shifted. The relative increase of the electron
densities always plays more important role in plasmon excitations than the
decrease of the symmetry of the QDs. Moreover, along the zigzag-edge
direction, though there are some small absorption peaks in the low-energy

b2227_P2-V1_Ch-03.indd 45 23-Jun-17 2:55:29 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

46  H. Yin

Figure 3. The dipole responses (optical absorptions) of the pyridine-like nitrogen-doped

GQDs to an impulse excitation polarized in the X-axis and Y-axis directions. The insets are
schematic diagrams of these QDs, respectively.42

b2227_P2-V1_Ch-03.indd 46 23-Jun-17 2:55:30 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Plasmons in Graphene Quantum Dots 47

Figure 4. Optical absorptions of the substitutional nitrogen-doped hexagonal GQDs to an

impulse excitation polarized in the X-axis and Y-axis directions. The insets are schematic dia-
grams of the substitutional nitrogen-doped hexagonal GQDs, respectively.42

resonance region, the main lower-energy absorption peak of substitutional

nitrogen-doped rectangular GQDs almost does not move. The results show
that, in different directions, substitutional nitrogen doping has different
effects on the plasmon resonance.
Using the time-dependent density functional theory, Yin et al. also
investigated NIR plasmon in N-doped hexagonal GQDs.43 Along a certain

b2227_P2-V1_Ch-03.indd 47 23-Jun-17 2:55:33 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

48  H. Yin

Figure 5. Optical absorptions of the substitutional nitrogen-doped rectangular GQDs to an

impulse excitation polarized in the X-axis and Y-axis directions. The insets are schematic dia-
grams of the substitutional nitrogen-doped rectangular GQDs respectively.42

direction, for N-doped hexagonal GQDs whose side length is 1 nm, there
are greater intensity plasmon resonances throughout the near-infrared spec-
tral region, as shown in Figure 6. The electrons which participate in these
near-infrared plasmon resonances oscillate back and forth between the
center and edge regions of the hexagonal QDs. The formation of near-
infrared plasmon resonance mode depends on nitrogen-doped position and
scale size of the GQD. It is only when the nitrogen-doped location is close
to the edge of the QDs that near-infrared plasmon resonance mode of the
GQD will be formed. For N-doped hexagonal GQDs whose side length is
less than 1 nm, there is no plasmon resonance in the near-infrared spectral
region.

3. Plasmons in GQD Dimers

Plasmonic nanoparticle pairs known as dimers have attracted much attention
in the past few years because they are important for understanding the

b2227_P2-V1_Ch-03.indd 48 23-Jun-17 2:55:34 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Plasmons in Graphene Quantum Dots 49

Figure 6. Optical absorption of different nitrogen-doped GQDs to an impulse excitation

polarized in the X-axis direction. The insets are schematic diagrams of different nitrogen-
doped GQDs.43

dynamics of hybridized plasmons in complex QDs.44 Coupled nanoparticle

pairs can give rise to very large field enhancements in their gap region due to
the high-charge densities induced at the opposite sides of the junction at
plasmon resonance. This opens a route to numerous practical applications,
such as surface-enhanced Raman scattering,45 nonlinear optics,46 fluorescence
amplication,47 and single-molecule sensing.48 As a general characteristic, for
larger separations, each nanoparticle remains neutral during a plasmon excita-
tion. The bonding dimer plasmon mode red-shifts monotonically with
decreasing interparticle separation. The quantum calculations agree with the
predictions of the classical approach for both plasmon energy and field
enhancement. As the interparticle distance is further reduced, in the crossover
regime, quantum mechanical effects start to be important, and electron tun-
neling across the junction can significantly reduce the electromagnetic inter-
actions between the two particles. When smaller gaps are a few angstroms, the
charge transfer plasmon mode appears which blue-shifts with decreasing
interparticle separation.43 Since localized surface plasmon resonance is sensi-
tively dependent on the geometry of the QD, various types of dimers struc-
tures have been investigated, such as ring and elliptical disk dimers, nanoshell
dimers, nanorod dimers,and triangular bowtie dimers.49
Yin et al. investigated plasmon excitations in GQD dimers by time-
dependent density functional theory.50 They found that, for larger

b2227_P2-V1_Ch-03.indd 49 23-Jun-17 2:55:34 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

50  H. Yin

Figure 7. Optical absorption of the hexagonal GQD and hexagonal GQD dimers to an
impulse excitation polarized in the X-axis directions.50

separations, the interaction between two GQDs is capacitive. In this situation,

when two GQDs approach to each other, low-energy plasmon modes of
GQD dimers show red-shift with the decrease of the gap, as shown in
Figure 7. When the gap distance is further decreased, because of the electrons
tunneling across the dimer junction, plasmon resonance modes of GQD
dimers are significantly modified, and the hybrid plasmon modes emerge. The
hybrid plasmon modes show further red-shift with the decrease of the gap.
The evolution of the plasmon resonance mode of GQD dimers does not
depend on the shape of the GQDs, as shown in Figures 7 and 8.
Using time-dependent density functional theory, Yin et al. also investi-
gated plasmon excitation and plasmon-induced electron transport in GQDs
connected by carbon atomic chains, as shown in Figure 9.51 With the increase
of the chain, in the low energy resonance region, absorption spectrum of the
system shows red-shift. For the longer carbon atoms chain, in the NIR and
mid-infrared spectrum region, system has a strong absorption. In the infrared
spectral region, plasmon resonance modes of GQDs connected by carbon
atomic chains also depend on the parity of atomic number in the chains.

b2227_P2-V1_Ch-03.indd 50 23-Jun-17 2:55:35 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Plasmons in Graphene Quantum Dots 51

Figure 8. Optical absorption of the rectangle GQD and rectangle GQD dimers to an
impulse excitation polarized in the X-axis directions.50

In the low energy spectrum area, induced current intensity of the greater
intensity plasmon resonance mode is also larger.
Thongrattanasiri et al. performed realistic, quantum-mechanical calcula-
tions of the optical response of graphene dimers formed by nanodisks and
nanotriangles, showing a strong sensitivity of the level of enhancement to the
type of carbon edges near the gap region with armchair edges favoring
stronger interactions than zigzag edges.52 Their quantum-mechanical descrip-
tion automatically incorporates non-local effects that are absent in classical
electromagnetic theory, leading to over an order of magnitude higher

b2227_P2-V1_Ch-03.indd 51 23-Jun-17 2:55:35 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

52  H. Yin

Figure 9. Optical absorption of GQDs connected by carbon atomic chains to an impulse

excitation polarized in the directions along the chain of carbon atoms. The variable number
represents the atom number in the carbon atom chain.51

enhancement in armchair structures. Moreover, Rosolen et al. investigated

the infrared response of graphene dimers with various doping and polariza-
tion configurations.53 The interaction between the plasmonic resonances of
graphene nanodisks leads to a rich, tunable behavior.

4. Conclusions
Surface plasmon of the graphene has become one of the world’s research
hotspots due to its novel optical properties. This section discusses the physical
nature of plasmon excitation which is modulated in GQDs and GQD dimers.
Compared with the plasmon in the macroscopic graphene, the plasmon in
GQDs has some different properties due to the effects of the size and the
dimensional confinement. In lower-energy resonance zone, the spectral band
is greatly broadened, and the photoabsorption strength line splits. Because of
the electromagnetic coupling between the QDs, GQD dimers exhibit differ-
ent optical properties. For plasmon regulation and control, these results pro-
vide a solid theoretical guidance.

References
1. Grigorenko AN, Polini M, Novoselov KS. Graphene plasmonics. Nat Photon 2012; 6:
749–758.

b2227_P2-V1_Ch-03.indd 52 23-Jun-17 2:55:36 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Plasmons in Graphene Quantum Dots 53

2. Jablan M, Buljan H, Soljačić M. Plasmonics in graphene at infrared frequencies. Phys Rev

B 2009; 80: 245435.
3. Fei Z, Rodin S, Andreev GO, Bao W, McLeod AS, Wagner M, Zhang LM, Zhao Z,
Thiemens M, Dominguez G, Fogler MM, Castro N, Lau CN, Keilmann F, Basov DN. Gate-
tuning of graphene plasmons revealed by infrared nano-imaging. Nature 2012; 487: 82–85.
4. Chen J, Badioli M, Alonso-Gonzalez P, Thongrattanasiri S, Huth F, Osmond J,
Spasenovic M, Centeno A, Pesquera A, Godignon P, Zurutuza EA, Camara N, de Abajo
FJG, Koppens HL. Optical nano-imaging of gate-tunable graphene plasmons. Nature
2012; 487: 77–81.
5. Papasimakis N, Luo Z, Shen ZX, De Angelis F, Di Fabrizio E, Nikolaenko AE, Zheludev
NI. Graphene in a photonic metamaterial. Opt Express 2010; 18: 8353–8359.
6. Echtermeyer TJ, Britnell L, Jasnos PK, Lombardo A, Gorbachev RV, Grigorenko AN,
Geim AK, Ferrari AC, Novoselov KS. Strong plasmonic enhancement of photovoltage in
graphene. Nat Commun 2011; 2: 458.
7. Fang N, Lee H, Sun C, Zhang X. Sub-diffraction-limited optical imaging with a silver
superlens. Science 2005; 308: 534–537.
8. Prodan E, Radloff C, Halas N J, Nordlander P. A hybridization model for the plasmon
response of complex nanostructures. Science 2003; 302: 419–422.
9. Nie S, Emory SR. Probing single molecules and single nanoparticles by surface-enhanced
Raman scattering. Science 1997; 275: 1102–1106.
10. Garcia MA. Surface Plasmons in metallic nanoparticles: Fundamentals and applications.
J Phys D Appl Phys 2012; 45: 389501.
11. Barnes WL, Dereux A, Ebbesen TW. Surface plasmon subwavelength optics. Nature
2003; 424: 824–830.
12. Fritzsche W, Taton TA. Metal nanoparticles as labels for heterogeneous, chip-based DNA
detection. Nanotechnology 2003; 14: R63–R73.
13. Pillai S, Catchpole KR, Trupke T, Green MA. Surface plasmon enhanced silicon solar cells.
J Appl Phys 2007; 101: 093105.
14. Narayanan R, El-Sayed MA. Catalysis with transition metal nanoparticles in colloidal solution:
Nanoparticle shape dependence and stability. J Phys Chem B 2005; 109: 12663–12676.
15. Ozbay E. Plasmonics: Merging photonics and electronics at nanoscale dimensions. Science
2006; 311: 189–193.
16. Wang W, Apell P, Kinaret J. Edge plasmons in graphene nanostructures. Phys Rev B 2011;
84: 085423.
17. Mishchenko EG, Shytov AV, Silvestrov PG. Guided plasmons in graphene p–n junctions.
Phys Rev Lett 2010; 104: 156806.
18. Sahu B, Min H, Banerjee SK. Edge saturation effects on the magnetism and band gaps in
multilayer graphene ribbons and flakes. Phys Rev B 2011; 84: 075481.
19. Yin HF, Zhang H. Plasmons in graphene nanostructures. J Appl Phys 2012; 111: 103502.
20. Zhang H, Yin HF, Zhang KB, Lin JH. Progress of surface plasmon research based on
time-dependent density functional theory. Acta Phys Sin 2015; 64: 077303.
21. Yin HF, Zhang H. Plasmon excitation in ring graphene nanostructures. Journal of
Sichuan University (Natural Science Edition) 2015; 52: 343–347.
22. Yan H, Low T, Zhu W, Wu Y, Freitag M, Li X, Guinea F, Avouris P, Xia F. Damping
pathways of mid-infrared plasmons in graphene nanostructures. Nat Photon 2013; 7:
394–400.

b2227_P2-V1_Ch-03.indd 53 23-Jun-17 2:55:36 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

54  H. Yin

23. Wang W, Li BH, Stassen E, Mortensen NA, Christensen J. Localized surface plasmons in
vibrating graphene nanodisks. Nanoscale 2016; 8: 3809–3815.
24. García de Abajo FJ. Graphene plasmonics: Challenges and opportunities. ACS Photon
2014; 1: 135–152.
25. Cox JD, García de Abajo FJ. Plasmon-enhanced nonlinear wave mixing in nanostructured
graphene. ACS Photon 2015; 2: 306−312.
26. Manjavacas A, Marchesin F, Thongrattanasiri S, Koval P, Nordlander P, Sánchez-Portal D,
García de Abajo FJ. Tunable molecular plasmons in polycyclic aromatic hydrocarbons.
ACS Nano 2013; 7: 3635−3643.
27. Manzoni MT, Silveiro I, García de Abajo FJ, Chang DE. Second-order quantum nonlin-
ear optical processes in graphene nanostructures. arXiv:1406.4360.
28. Cox JD, García de Abajo FJ. Electrically tunable nonlinear plasmonics in graphene nanois-
lands. Nat Commun 2014; 5: 5725–5732.
29. Schiros T, Nordlund D, Pálová L, Prezzi D, Zhao L, Kim KS, Wurstbauer U, Gutiérrez
C, Delongchamp D, Jaye C, Fischer D, Ogasawara H, Pettersson L, Reichman DR, Kim
P, Hybertsen MS, Pasupathy AN. Connecting dopant bond type with electronic structure
in N-doped graphene. Nano Lett 2012; 12: 4025–4031.
30. Wang Y, Shao Y, Matson DW, Li J, Lin Y. Nitrogen-doped graphene and its application
in electrochemical biosensing. ACS Nano 2010; 4: 1790–1798.
31. Lee SU, Belosludov RV, Mizuseki H, Kawazoe Y. Designing nanogadgetry for nanoelec-
tronic devices with nitrogen-doped capped carbon nanotubes. Small 2009; 5:
1769–1775.
32. Zhao L, He R, Rim KT, Rim KT, Kim KS, Zhou H, Gutierrez C, Chockalingam S,
Arguello C, Palova L, Reichman DR, Heinz TF, Kim P, Pinczuk A, Flynn GW,
Pasupathy AN. Visualizing individual nitrogen dopants in monolayer graphene. Science
2011; 333: 999–1003.
33. Gao H, Song L, Guo W, Huang L, Yang D, Wang F, Zuo Y, Fan X, Liu Z, Gao W, Vajtai
R, Hackenberg K, Ajayan PM. A simple method to synthesize continuous large area
nitrogen-doped graphene. Carbon 2012; 50: 4476–4482.
34. Li M, Wu W, Ren W, Cheng H, Tang N, Zhong W, Du YW. Synthesis and upconversion
luminescence of n-doped graphene quantum dots. Appl Phys Lett 2012; 101: 103107.
35. Guo B, Liu Q, Chen E, Zhu H, Fang L, Gong JR. Controllable N-doping of graphene.
Nano Lett 2010; 10: 4975–4980.
36. Xiang HJ, Huang B, Li ZY, Wei SH, Yang JL, Gong XG. Ordered semiconducting nitrogen-
graphene alloys. Phys Rev X 2012; 2: 011003.
37. Ritter KA, Lyding JW. The influence of edge structure on the electronic properties of
graphene quantum dots and nanoribbons. Nat Mater 2009; 8: 235–242.
38. Yan X, Cui X, Li B, Li L. Large solution-processable graphene quantum dots as light
absorbers for photovoltaics. Nano Lett 2010; 10: 1869–1873.
39. Yan X, Cui X, Li L. Synthesis of large, stable colloidal graphene quantum dots with tun-
able size .J Am Chem Soc 2010; 132: 5944–5945.
40. Li Y, Zhao Y, Cheng H, Hu Y, Shi G, Dai L, Qu L. Nitrogen-doped graphene quantum
dots with oxygen-rich functional groups. J Am Chem Soc 2012; 134: 15–18.
41. Jin SH, Kim DH, Jun GH, Hong SH, Jeon S. Tuning the photoluminescence of graphene
quantum dots through the charge transfer effect of functional groups. ACS Nano 2013;
7: 1239–1245.

b2227_P2-V1_Ch-03.indd 54 23-Jun-17 2:55:36 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Plasmons in Graphene Quantum Dots 55

42. Yin HF, Chen G, Xiang G, Zhang H. Plasmon excitation in nitrogen-doped graphene
nanostructures. Chin J Lumin 2014; 35: 1297–1306.
43. Yin HF, Zhang H,Yue L. Near-infrared plasmon study on N-doped hexagonal graphene
nanostructures. Acta Phys-Chim Sin 2014; 30: 1049–1054.
44. Zuloaga J, Prodan E, Nordlander P. Quantum description of the plasmon resonances of
a nanoparticle dimer. Nano Lett 2009; 9: 887–891.
45. Lim DK, Jeon KS, Kim HM, Nam JM, Suh YD. Nanogap-engineerable Raman-active
nanodumbbells for single-molecule detection. Nat Mater 2010; 9: 60–67.
46. Kim S, Jin J, Kim YJ, Park IY, Kim Y, Kim SW. High-harmonic generation by resonant
plasmon field enhancement. Nat 2008; 453: 757–760.
47. Giannini V, Fernández-Domínguez AI, Heck SC, Maier SA. Plasmonic nanoantennas:
fundamentals and their use in controlling the radiative properties of nanoemitters. Chem
Rev 2011; 111: 3888–3912.
48. Liu N, Tang ML, Hentschel M, Giessen H, Alivisatos AP. Nanoantenna-enhanced gas
sensing in a single tailored nanofocus. Nat Mater 2011; 10: 631–637.
49. Halas NJ, Lal S, Chang WS, Link S, Nordlander P. Plasmons in strongly coupled metallic
nanostructures. Chem Rev 2011; 111: 3913–3961.
50. Yin HF, Zhang H, Liu D. Plasmon excitation in graphene quantum dot dimers. Journal
of Sichuan Normal University (Natural Science) 2014; 37: 691–696.
51. Yin HF, Zeng CH, Yue L. Plasmon excitation in graphene quantum dots connected by
carbon atomic chains. Journal of University of Jinan (Science and Technology) 2016; 30: 1–6.
52. Thongrattanasiri S, Javier Garcı´a de Abajo F. Optical field enhancement by strong plas-
mon interaction in graphene nanostructures. Phys Rev Lett 2013; 110: 187401.
53. Rosolen G, Maes B. Asymmetric and connected graphene dimers for a tunable plasmonic
response. Phys Rev B 2015; 92: 205405.

b2227_P2-V1_Ch-03.indd 55 23-Jun-17 2:55:36 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Chapter 4

Application of Graphene Quantum Dots in

Medical and Pharmaceutical Analyses

Xiaolei Zhang*, Jing Wang and Gongjun Yang#

China Pharmaceutical University, Nanjing 211198, P.R. China

*[email protected]; #[email protected]

Nanotechnology has been widely applied in nanomedicine for the purpose of

molecular level medical research such as diagnostic, prevention, and therapy to
improve medical skill in recent years. Toward this purpose, different kinds of sensors
based on nanoparticles have been constructed due to their high-sensitivity, specificity
and rapidity in analysis.
This review makes a latest overview of the emerging chemical sensors and
biosensors based on graphene quantum dots (GQDs) or composite materials
of GQDs, which can be mainly employed for the detection of cancer biomarkers,
other disease biomarkers and pharmaceutical components, and the screening of
drug molecules. More and more such sensors have shown the potential for the early
diagnosis of disease, tracking disease treatment, mechanism of drug action studying
and drug choice in the clinical area.

1. Introduction
Graphene quantum dots (GQDs) have attracted extensive attention of scien-
tists owing to their excellent chemical/physical qualities arising from the
pronounced quantum confinement and edge effects. In contrast to other
semiconductor quantum dots and organic dyes, GQDs show low cytotoxicity,
good biocompatibility, stable photoluminescence (PL), high electron mobil-
ity, better surface grafting, and so on, thus making them promising in opto-
electronic devices, sensors, bioimaging, etc.1–3 At present, applications of

57

b2227_P2-V1_Ch-04.indd 57 23-Jun-17 2:56:01 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

58  X. Zhang, J. Wang & G. Yang

GQDs have much rapid development in pharmaceutical determination, clini-

cal diagnosis, drug delivery, cancer cell imaging as well as cancer therapy in
the field of medicine. In the context, we mainly discuss the biosensor and
chemical sensor based on GQDs for medical and pharmaceutical analyses.

2. Application in Medical Analysis

It has been known that several ions are of serious concern to human health.
There have been great benefits for clinical diagnosis and therapy through
accurate and sensitive analysis of those anions and cations in the living organ-
ism. There have also been diverse sensors based on GQDs fabricated for the
determination of ions,4,5 including Al3+,6 Au3+,7 Cl–,8 CN–,9 Cr6+,10,11 Fe3+,12,13
Cu2+,14 Hg2+,15-18 and Pb2+.19-21
Pyrophosphate ion (PPi) is an important biological metabolite of ATP
hydrolysis and DNA polymerization. The previous studies revealed that PPi
level is related to the pathological processes of arthritis. Liu et al.22 designed
a facile and effective switch-on fluorescent strategy based on N and S
co-doped GQDs (N–S/GQDs) for monitoring pyrophosphate ions in syno-
vial fluid of arthritis patients. The mechanism about the sensing strategy of
PPi by using N–S/GQDs combined with Fe3+ was as follows. Firstly, the addi-
tion of Fe3+ quenched N–S/GQDs fluorescent intensity. Secondly, the addi-
tion of PPi into the N–S/GQD solution allowed the florescence to
substantially recover from quenching. The quantitative basis is according to
the relationship between the recovered fluorescence intensities and concentra-
tions of PPi.
At present, more and more biomolecules have been chosen as biomarkers
for diagnosis of various diseases. Application of GQDs in biomolecules detec-
tion for medical studies would be introduced below.

2.1. Adenosine triphosphate

It is well known that adenosine triphosphate (ATP) stores and transfers
chemical energy as “energy currency” in the most animate beings. The detec-
tion of ATP is of clinical importance since the concentration and dissipative
rate of ATP is related to many diseases such as hypoxia, hypoglycemia,
ischemia, Parkinson’s disease and some malignant tumors. Lu et al.23 devel-
oped a newly anodic electrochemiluminescence (ECL) aptasensor based on
water-soluble GQDs with H2O2 as coreactant for ATP detection. As shown
in Figure 1, the monodispersed SiO2 nanospheres were used as carriers for
GQDs and ssDNA2 was immobilized on the surface of SiO2/GQDs to form

b2227_P2-V1_Ch-04.indd 58 23-Jun-17 2:56:01 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Application of GQDs in Medical and Pharmaceutical Analyses 59

Figure 1. Schematic representation of the ECL aptamer ATP sensor. Reprinted with
permission from Ref. [23]. Copyright © 2013 Elsevier B.V.

SiO2/GQDs/ssDNA2 detection probe. The ssDNA1 was absorbed onto the

precleaned Au electrode and further treated with MCH (6-mercapto-1-­
hexanol) to obtain a well-aligned DNA monolayer. In the presence of ATP,
two fragments hybridized with each other, so SiO2/GQDs were caused to be
immobilized onto the electrode surface, resulting in ECL signal. Bright blue
fluorescent glutathione-functionalized GQDs (GQDs@GSH) can be used as
a probe to estimate the ATP level in cell lysates and human blood serum.24 In
the research, Fe3+ could quench the strong fluorescence of the GQDs@GSH
through chelation reaction. ATP has been found to have a high-affinity for
iron ions through Fe–O–P bonds. Therefore, the fluorescence signal could be
observed again on the addition of ATP owing to the release of GQDs@GSH.

2.2. Amino acids and biothiol

Biothiol compounds play important roles in maintaining proper body func-
tions, including glutathione, cysteine, and homocysteine. Amino acids also
play important roles in various biological progresses. Abnormal biothiols or
amino acids levels have been used for disease diagnosis and clinical
therapies.
Wu et al.25 reported a GQD-based method for the highly sensitive and
selective sensing of biothiols. Hg2+ was chosen to quench fluorescence of
GQDs via electron or energy transfer from the GQDs to Hg2+ ions in the
formation of Hg2+–GQD complex. Nonetheless, a biothiol compound that
selectively bond Hg2+ through Hg–S bonding interactions could recover the
fluorescence. That is to say the Hg2+–GQD complex dissociated and a

b2227_P2-V1_Ch-04.indd 59 23-Jun-17 2:56:01 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

60  X. Zhang, J. Wang & G. Yang

Figure 2. Schematic illustration of the mechanism of the GQD-based biothiol sensing.

Reprinted with permission from Ref. [25]. Copyright © 2013 Elsevier B.V.

fluorescence signal turned on again (Figure 2). The intensity enhancement

of the GQDs could be directly related to the amount of biothiol added to the
assay solution. It was reported that there were many kinds of amino acids and
biothiols testing were based on off–on PL mechanism, namely PL of GQDs
quenching by metal ions and turning on by analytes. Liu et al.26 also used
Hg2+ to quench fluorescence of N-doped GQDs for cysteine detection.
Eu-decorated GQDs system has been developed for the determination of
Cu2+ and L-cysteine.27 Eu3+ and GQDs can be employed to test glutamic acid
and aspartic acid.28 Au@Ag nanoparticles and GQDs grafted with polyethyl-
eneimine have been combinedly used for the detection of glutathione,
cysteine and homocysteine.29
In addition to the above methods for the detection of amino acids and
biothiols, there are some other different methods that emerged for the same
purpose. Ju et al.30 designed a novel Ag NP-N-GQDs–TMB–H2O2 colori-
metric sensor for the sensitive detection of glutathione (GSH). The Ag
NP-N-GQDs nanocomposite was successfully prepared via a simple photo-
chemical process. Then the TMB (3,3′,5,5′-tetramethylbenzidine), Ag
NP-N-GQDs and H2O2 were mixed and heated to 37°C, and held for 15
min. Due to the intrinsic peroxidase-like activity of Ag nanoparticle which
was to catalyze the oxidation of TMB to oxTMB with the presence of H2O2,
the solution showed bright blue color with a maximum absorbance at 652
nm. On the addition of GSH, the UV–vis absorption intensity decreased
because of catalytic reduction reaction between oxTMB and GSH, which was
the basis of quantification detection of GSH. L-cysteine (L-cys) also can be
determined by electrochemical sensor. Wang et al.31 developed a Prussian
Blue (PB) nanocomposite hybrid film anchored on a GQDs modified graph-
ite felt (GF) electrode for enhancing reaction rate of the PB formation.
A polypyrrole (PPy) film was electrodeposited on the surface of the electrode
to form PPy/GQDs@PB/GF electrode. At potential of 0.22 V, a good linear
relationship between the oxidation current and concentration of L-cys has

b2227_P2-V1_Ch-04.indd 60 23-Jun-17 2:56:01 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Application of GQDs in Medical and Pharmaceutical Analyses 61

been obtained. Moreover, Ou et al.32 reported an electrochemical chiral inter-

face for the recognition of tryptophan isomers by the nanocomposites of
GQDs and β-cyclodextrin.

2.3. Nuclei acids

MicroRNAs (miRNAs) are a class of non-coding RNA molecules, playing
critical regulatory functions in many biological processes such as tumorigen-
esis, metastasis, diagnosis, prognosis, cell differentiation, cell apoptosis and
protein synthesis. The previous studies on functions of miRNA proved that
it could be an oncogene or a tumor suppressor gene. Therefore, miRNAs
analysis methods have shown great potential in basic and clinical research on
tumor. As shown in Figure 3, Zhang et al.33 prepared boron-doped GQDs
tagged DNA as ELC probe which dropped on the surface of AuNPs modi-
fied Pt electrode through S–Au bond to form Pt/Au/B-GQDs-DNA elec-
trode. After hybridization with the target miRNA-20a, the ECL intensity of
Pt/Au/B-GQDs-DNA/miRNA-20a was decreased. The reason was that the
folded structure of the hairpin DNA was transformed into a linear double
helix after hybridization of probe DNA with the target miRNA-20a. So, it
led the B-GQDs tag to remove away from the electrode surface that impeded
the ECL reaction between B-GQDs and K2S2O8 (coreactant), causing the
decrease of ECL probe efficiency. It was confirmed that the sensor was
­feasible to be applied to analyze the concentrations of miRNA-20a in extra-
cellular secretion.

Figure 3. Schematic illustration of the preparation processes of ECL electrode and the detec-
tion procedures of the ECL sensor. Reprinted with permission from Ref. [33]. Copyright ©
2015 Elsevier Ltd.

b2227_P2-V1_Ch-04.indd 61 23-Jun-17 2:56:02 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

62  X. Zhang, J. Wang & G. Yang

Another kind of miRNA-155 was sensitively detected by a simple electro-

chemical RNA biosensor from 1 fmol/L to 100 pmol/L with a detection
limit of 0.14 fmol/L.34 Firstly, the capture DNA was fixed onto a 2-mm
diameter gold working electrode (GE). Secondly, part of target RNA hybrid-
ized with capture DNA and NH2-DNA hybridized with another part of RNA
in the presence of miRNA-155. Thirdly, the activated carboxyl group of
GQDs reacted with the amino of NH2-DNA and then non-covalently bound
to horseradish peroxidase (HRP). Finally, H2O2 oxidized the colorless TMB
into the blue derivative of oxTMB, resulting in the electrochemical reduction
current of TMB.
Similarly, sensitive, effective and rapid detection of specific DNA is critical
in medical diagnosis, gene therapy, and sensing of genetic disorders. For
instance, GQDs were modified readily onto the surface of pyrolytic graphite
(PG) electrode by Zhao et al,35 and then the probe ssDNA (ssDNA-1) was
easily immobilized through the interaction between the nucleobases and
­graphene (Gr). The immobilized ssDNA-1 would inhibit the electron transfer
between the electro-active species [Fe(CN)6]3-/4- and the electrode because
of the electrostatic repulsion. Nevertheless, in the presence of the target mol-
ecules (ssDNA-2), electrochemical response signal obtained at the electrode
changed, which was because of the fact that two complementary ssDNA can
hybridize with each other to form a double helix structure, resulting in the
immobilized ssDNA-1 being removed from the surface of the electrode. The
fabricated electrochemical biosensors have been applied for the detection of
thrombin. Qian et al.,36,37 designed a series of high-selective and sensitive
fluorescent nanosensor for DNA detection based on biocompatible GQDs
and carbon nanotubes. Specific fluorescence “on–off–on” process was the
critical signal change procedure in detection, which was based on base pairing
specificity of DNA and unique fluorescence resonance energy transfer (FRET)
between GQDs and CNTs. To put it simply, single-stranded DNA probe
(ssDNA–rGQDs) that exhibits strong blue fluorescence was adsorbed on the
surface of CNTs through electrostatic attraction and π–π stacking interaction.
Such interactions would cause fluorescence quenching. While target DNA
hybridized with ssDNA that broke up electrostatic attraction and π–π stacking
interaction between ssDNA–rGQDs and CNTs, the fluorescence reoccurred.
The proposed principle was employed for simultaneous detection of multiple
DNA targets to bring new opportunities for improving the accuracy of early
disease detection. Dual-color GQDs were employed to synthesize blue probe
(P1) and green probe (P2) for recognition of two DNA targets through two
cycles of fluorescence on–off–on respectively.

b2227_P2-V1_Ch-04.indd 62 23-Jun-17 2:56:02 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Application of GQDs in Medical and Pharmaceutical Analyses 63

Figure 4. Schematic illustration of NEase-assisted target recycling and procedures of ratio-

metric fluorescence assay. Reprinted with permission from Ref. [38]. Copyright © 2016
Elsevier B.V.

Here, some researchers focus on introducing a GQDs-based ratiometric,

label-free and amplified fluorescence strategy for the detection of DNA by
coupling the cascade amplification of NEase and DNAzyme developed by
Wang et al.38 As shown in Figure 4, the hairpin DNA probe is composed of
three domains, the G-riched DNAzyme sequence (pink region) that is
hybridized with complementary purple region, and the green region that is
designed to recognize the target DNA. When hybridization occurred with a
perfectly matched target DNA, the hairpin DNA was opened. Then the
Nicking endonuclease (NEase) specifically cleaved the hairpin probe into two
fragments. The released target could immediately hybridize with a new hair-
pin DNA and the next cycle of cleavage started. So a lot of G-riched
DNAzyme sequences were released by this means. Upon the addition of
hemin, the G-riched DNAzyme sequences would interact with hemin to form
the activated G-quadruplex/hemin DNAzyme. After removing redundant
free hemin by SWCNTs absorption, o-phenylenediamine (OPD) was oxidized
by the catalyst of G-quadruplex/hemin DNAzymes with the aid of H2O2.
The oxidized product DAP quenched the fluorescence intensity of GQDs (at
460 nm), accompanied with the emergence of a new emission of DAP (at 564
nm). It was proved that the fluorescence intensity ratio of I564/I460 increased
linearly with the logarithmical concentration of target DNA. In this research,
compared to the commonly used single intensity-based assay, ratiometric

b2227_P2-V1_Ch-04.indd 63 23-Jun-17 2:56:02 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

64  X. Zhang, J. Wang & G. Yang

detection demonstrated the advantage of sensitivity and accuracy, which can

effectively rule out high-background interference or false positive signals.

2.4. Antigen
Certain antigens such as specific antigen and carcinoembryonic antigen (CEA)
have been proved to be the indicator of diseases. Accurate analysis of such
antigen plays an important role in early detection and therapy monitoring.
Heart attack (myocardial infarction) is one of the major reasons of death
among people suffering from cardiovascular diseases. Cardiac Troponin I
(cTnI) marker has been considered as the “gold standard” for determining
heart attack in patients. Thus, cTnI level in blood serum is directly associated
with death risk from heart attack. Bhatnagar et al.39 reported a simple, rapid
and sensitive FRET — based immunosensor for detection of cTnI using
GQDs and Gr. The mechanism of immunosensing was shown in Figure 5. The
anti-cTnI/af-GQDs nanoprobe was constructed with monoclonal antibody
cardiac Troponin I (anti-cTnI) and amine-functionalized graphene quantum
dots (af-GQDs) by covalent conjugation which absorbed UV radiations

Figure 5. Schematic representation of bioconjugation of anti-cTnI with af-GQDs and

mechanism of immunosensing. Reprinted with permission from Ref. [39]. Copyright © 2015
Elsevier B.V.

b2227_P2-V1_Ch-04.indd 64 23-Jun-17 2:56:02 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Application of GQDs in Medical and Pharmaceutical Analyses 65

(360 nm) and emitted blue radiations (437 nm). The fluorescence intensity
of the nanoprobe was quenched after the adsorption of anti-cTnI/af-GQDs
on the surface of Gr through electrostatic attraction and π–π stacking interac-
tion. Finally, the addition of target antigen (cTnI) resulted in fluorescence
recovery. The reason is that due to the specific antibody–antigen interaction,
the detachment of π–π interaction between GQDs conjugate and Gr sheet
occurs and then hinders the FRET process. The good linear relationship
between PL intensity (I) and logarithm of cTnI concentrations was obtained
in the range of 0.001–1000 ng/mL with a limit of detection of 0.192 pg/
mL. Therefore, the proposed immunosensor based on GQDs was founded to
apply in the early stage of myocardial infarction causing heart attack.
Zou et al.40 designed a novel dual-mode immunoassay to detect a tuber-
culosis (TB) antigen, CFP-10. They produced one-dimensionally aligned
magnetoplasmonic (MagPlas) nanowires (NWs), i.e. Fe3O4@PEI@Au nano-
particles as sensing platform which were aligned on the surfaces of silicon
wafers under the external magnetic field. Then GBP-anti CPF10 [G2] and
GBP-anti CPF10 [G3] antibodies were fixed separately on the surfaces of the
MagPlas NPs and GQDs. The target antigen (CFP10) bound with both anti-
bodies via the antibody−antigen interaction to form sandwich-type nanocom-
plex. The nanocomplex with specific antigens sent out stronger SERS signals
than those with no specific antigen. The proposed method was expected to
be further applied in high-throughput screening of target molecules.
Prostate-specific antigen (PSA) was detected with a fluorescent turn-on
nanoprobe by Pei et al.41 The GQDs@Ag core-shell nanocrystals were synthe-
sized and attached with detection anti-PSA antibodies (Ab2) to form the
immunosensing probe (GQDs@Ag–Ab2). Capture anti-PSA antibodies (Ab1)
were directly adsorbed onto magnetic beads (MBs) to form capture probe
(MBs–Ab1). A sandwich type of immunocomplex with PSA, MBs–Ab1 and
GQDs@Ag–Ab2 was constructed and then separated using an external mag-
net. Finally, when Ag shell was etched using H2O2, the incorporated GQDs
were released and the fluorescence of the GQDs was recovered.
Zhang et al.42 developed a novel strategy for ultrasensitive ECL
­sandwich-type immunosensor based on the green-luminescence N-GQDs
oxidation using AuNFs modified paper as working electrode (Au-PWE) to
detect α-fetoprotein antigen (AFP). In this work, an interconnected gold
nanoflowers (AuNFs) layer was grown on the surfaces of the paper in order
to fabricate paper working electrode (PWE) and Ab1 was immobilized on
the Au-PWE. MWCNTs/N-GQDs/Ab2 labels were prepared by the
method that N-GQDs were absorbed on the surface of MWCNTs and then
Ab2 was immobilized on the MWCNTs/GQDs composites. Only when

b2227_P2-V1_Ch-04.indd 65 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

66  X. Zhang, J. Wang & G. Yang

adding of AFP, a strong anodic ECL signal could be observed due to sand-
wich immune complex formation through immunoreaction among Ab1,
AFP and Ab2. Li et al.43 fabricated a highly sensitive three-dimensional (3D)
origami device combined with ECL immunosensor for sensitive point-of-
care testing of CEA. In this sensing system, nanoporous gold/chitosan
modified PWE was used as sensor platform and GQDs functionalized Au@
Pt core–shell nanoparticles (GQDs/Au@Pt) were designed as signal labels.
The role of GQDs, gold nanoflowers and gold nanoparticles employed in
above two types of sensors was to amplify ECL signal and further improve
the sensitivity.

2.5. Trypsin
Trypsin, which is highly correlated with the occurrence inflammation and
related to the onset of pancreatitis, is a pancreatic digestive enzyme. Thus, it
is very important for the clinical detection of trypsin in urine samples for self-
checking by the suspect patient. Poon et al.44 selected GQDs as signal donor
and coumarin fluorophore as an acceptor for the detection of trypsin urine.
BSA was chosen for the linking of the donor and acceptor. The florescence
quenched as a result of FRET when the donor and acceptor were in close
proximity after being linked. While in the presence of trypsin, BSA was
cleaved into small fragments and the florescence recovered. On the same
principle, Li et al.45 demonstrated a novel fluorescent biosensor for trypsin.
GQDs were self-assembled in the presence of cytochrome c (Cyt c) and then
the florescence quenched. However, trypsin cleaved the peptide bonds of Cyt
c to give lysine and arginine residues, both of them induced a subsequent
increase in GQDs florescence intensity.

2.6. Dopamine
Dopamine (DA) is a neurotransmitter that plays an essential role in the func-
tion of the central nervous and cardiovascular systems. The dysfunctions of
DA are related to several important nervous system diseases, such as
Parkinson’s disease, schizophrenia and anorexia. Facile and accurate analysis
of DA could be good for clinical diagnosis of special neurological diseases.
GODs were used mainly as fluorescent probe with the same turn-off
­fluorescent strategy for the analysis of DA. For example, DA46 could be
adsorbed on the surface of GQDs and became pDA (polydopamine) through
the self-polymerization effect of DA in alkaline condition, which resulted in
an efficient fluorescence quenching of GQDs. In the same way,

b2227_P2-V1_Ch-04.indd 66 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Application of GQDs in Medical and Pharmaceutical Analyses 67

dopamine–quinine, oxidization product of DA by ambient O2 in alkaline

solution, could effectively quench fluorescence of GQDs.47 Xi Zhou et al.48
synthesized polypyrrole/GQDs core/shell hybrids which may lead to
enhancement of fluorescence intensity of GQDs by surface passivation. When
DA molecules effectively and specifically were absorbed onto the surface of
polypyrrole/GQDs composites through hydrogen bonds between DA and
PPY (­ polypyrrole), the fluorescence intensities of GQDs were quenched.
Moreover, the GQDs were modified on the surface of glassy carbon electrode
(GCE) through covalent self-assembled method based on amide covalent
bond formed between GQDs and ethylenediamine.49 The obtained modified
electrode exhibited better electrochemical response to DA than normal
­
bare electrode.
Peroxidase-like catalytic activity of GQDs has been widely used for H2O2
and glucose detection.50–53 Narsingh R. Nirala et al.54 reported a colorimetric
detection of free cholesterol method based on highly efficient peroxidase
mimetic activity of GQDs for the first time. Cholesterol is an important part
of animal cell membranes. However, when the amount of cholesterol exceeds
the amount required, pathological accumulations of cholesterol in blood ves-
sels can develop, resulting in atherosclerosis. H2O2 is the main product of
cholesterol oxidation by cholesterol oxidase (ChOx) in the presence of oxy-
gen. Peroxidase substrate TMB can be oxidized to produce a blue colored
product oxTMB in the presence of H2O2 with the assistance of GQDs cata-
lytic activity. Then the concentration of cholesterol could be determined
based on UV–vis intensity of oxTMB. The proposed method also showed
high-selectivity for cholesterol detection without being interfered by uric
acid, urea, ascorbic acid, cysteine and glucose.
Monitoring of uric acid in human plasma and urine is of great importance
in the physiological investigations and disease diagnosis since high level of
serum urate causes an inflammatory reaction, gouty arthritis. Amjadi et al.55
reported on the chemiluminescence (CL) of GQDs induced by direct chemi-
cal oxidation with Ce (IV). The CL emission spectrum of GQDs-Ce (IV)
system appeared a maximum at 470 nm in the range of 400–600 nm. It was
found that trace amount of uric acid has a diminishing effect on the reaction
due to the competition of uric acid with GQDs for Ce (IV). The linear rela-
tionship was found between the CL response and the concentration of uric
acid in the range of 1.0 × 10-6 –5.0 × 10-4 mol/L with a limit of detection
(3s) of 5.0 × 10-7mol/L.

b2227_P2-V1_Ch-04.indd 67 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

68  X. Zhang, J. Wang & G. Yang

3. Application in Pharmaceutical Analysis

GODs is not only applied for clinical disease diagnostic but also for drug
discovery and therapy. Protein kinases can catalyze the phosphorylation of
proteins by transferring phosphate groups to specific amino acids, which play
a critical regulatory role in many fundamental biological processes. Abnormal
expression of protein kinase has been implicated in a number of diseases
including cancers, HIV, carney complex, and so on. Wang et al.56 designed a
simple and sensitive PL assay method for the activity of casein kinase II
(CK2) identification and inhibitor screening. Peptide-modified GQDs bio-
conjugates (peptide-GQDs) were phosphorylated catalytically by CK2 in the
presence of ATP. After titration of Zr4+ ions into the solution of phosphoryl-
ated peptide-GQDs, PL was quenched owing to energy-transfer processes
resulted from aggregation of the GQDs triggered by Zr4+ ion coordination
with the phosphate groups on the peptide-GQDs. However, if CK2 was pre-
treated with different amounts of ellagic acid used as a potent CK2 inhibitor
in advance, the PL intensity increased with increasing ellagic acid concentra-
tion. Three other inhibitors (DRB, emodin, and quercetin) were also tested
in the identical concentrations with ellagic acid by this proposed method in
human serum. The result showed that the inhibition efficiency of ellagic acid
was the greatest through the comparison of PL intensities with each other.
So, it is possible to realize high-throughput screening for protein kinase
inhibitors and drug development in biological fluids. ECL assay based on
GQDs was also reported for protein kinase inhibitors screening. Liang et al.57
prepared anti-phosphoserine antibody conjugated graphene oxide (Ab-GO)
nanocomposite and peptide-GQDs electrode. The phosphorylated peptide-
GQDs modified electrode in the present of CK2 and ATP could capture
Ab-GO, resulting in ECL from the GQDs quenching. Likewise, Jinquan Liu
et al.58 modified GQDs with substrate peptide of target protein kinase A
(PKA) and functionalized AuNPs with the phosphorylated DNA and
G-quadruplex–hemin DNAzyme. In the presence of PKA, the peptide on the
GQDs was phosphorylated and the phosphorylated linker DNA on the sur-
face of AuNPs was integrated with the phosphorylated peptide by Zr4+. The
G-quadruplex–hemin DNAzyme on the surface of AuNPs could catalyze the
reduction of coreactant of H2O2. In both cases, the ECL intensity decreased
significantly. Of the two sensing systems, the relative protein kinase inhibitors
could be tested, which would be expected for inhibitor screening in drug
development.
Enhanced GQDs fluorescence nanosensor for acetylcholinesterase
­inhibitor screening has been proposed by Tian He et al.59 In Figure 6, the

b2227_P2-V1_Ch-04.indd 68 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Application of GQDs in Medical and Pharmaceutical Analyses 69

Figure 6. Schematic illustration of the assay strategy for the detection of acetylcholinesterase
activity and inhibitor screening. Reprinted with permission from Ref. [59]. Copyright © 2015
Elsevier B.V.

fluorescence of GQDs enhanced with EDC/NHS in the acetylcholinesterase

(AChE) immobilization procedure was quenched by the Hg2+ ion.
Acetylthiocholine iodide (ATCh) could be hydrolyzed by AChE to yield thio-
choline that interacted with Hg2+ to recover the quenched fluorescence.
Nevertheless, the fluorescence recovery could be restricted when an inhibitor
was simultaneously added, owing to activity of enzyme decreasing.
Most studies have demonstrated that a lot of kinds of drugs are likely to
be carcinogenic and have potential lethal side effects on human beings. It is
urgent to determine the concentration of drug to meet the demand of drug,
food and environmental safety. Meanwhile, close monitoring and determina-
tion of drug is much important for its clinical application.
Chloramphenicol (CAP) has been assayed with a label-free photoelectro-
chemical (PEC) aptasensor based on nitrogen-doped GQDs (N-GQDs).60
Liu et al. selected N-GQDs as visible light responsive materials to improve
photon-to-electricity conversion efficiency and designed a low-cost label-free
DNA aptamer for CAP as a biorecognition element. The aptamer could be
immobilized facilely on N-GQDs through π–π stacking interaction between

b2227_P2-V1_Ch-04.indd 69 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

70  X. Zhang, J. Wang & G. Yang

the nucleobases and N-GQDs to form aptamer/N-GQDs/FTO (F-doped

SnO2) electrode. Upon the addition of different amounts of CAP, the photo-
current response of aptamer/N-GQDs/FTO electrode increased with
increase in the concentrations of CAP due to the capture of more CAP mol-
ecules participating in the PEC process.
Fluoroimmunoassay based on regulation of the interaction between gra-
phene (Gr) and GQDs has been interpreted in the section of medical analysis
application. Detection of human immunoglobulin G (IgG) as well employed
such a luminescence resonance energy transfer (LRET) mechanism.61 In this
case, Gr was chosen as an acceptor and mouse anti-human immunoglobulin
G (mIgG, antibody)-conjugated GQDs were as donors. Owing to both the
π–π stacking interaction between Gr and GQDs and the non-specific binding
interaction between mIgG and the Gr surface, the luminescence quenching
of GQDs based on LRET occurred. Human IgG would bind the mIgG due
to the specific antibody–antigen interaction. So, a restoration of luminescence
could be observed, resulting from the increased distance between mIgG–
GQDs and Gr surface.
GQDs can be decorated with CdS doped GO sheets to develop a nano-
hybrid as initiator and platform for designing of nimesulide imprinted poly-
mer. Patra et al.62 firstly modified GO with dithiocarbamate moiety to make
a nanoiniferter and then prepared CdS-doped GO nanomaterial (CdS:GO).
The prepared CdS:GO was further combined with GQDs to develop a nano-
hybrid with enhanced fluorescence property. These nanohybrids were used
simultaneously as immobilization surface of nimesulide imprinted polymer. In
the fabrication of imprinted polymer matrix, N-vinyl caprolactam and vinyl
derivative of cystine were used as functional monomers, nimesulide was used
as a template molecule. After polymerization, the template was extracted
from resulting polymer matrix by 0.1 mol/L NaOH in dynamic mode. It was
observed that the rebinding of target molecule with polymer could quench
the fluorescence intensity because nimesulide did not show any fluorescence
signal in this range. After addition of nimesulide, the differential fluorescence
intensity value with fluorescence intensity of molecularly imprinted polymer
as baseline value was appropriate to the concentration of nimesulide. The
prepared optical sensor has the potential to detect nimesulide in human
blood, serum, plasma and urine samples without any interference effect.
Li et al.63 synthesized water-soluble multipositively charged dendrimeric
nanoparticles, i.e. guanidine-terminated dendrimer (PAMAM-Gu+) with
128 guanidinium ions (Gu+) distributed on the molecular surface.
PAMAM-Gu+ could bind to the carboxyl group of GQDs via electrostatic

b2227_P2-V1_Ch-04.indd 70 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Application of GQDs in Medical and Pharmaceutical Analyses 71

interaction, leading to the aggregation of GQDs, which resulted in the

degression of the fluorescence intensity. Heparin or chondroitin sulfate is
negatively charged that they could combine with PAMAM-Gu+ due to their
strong electrostatic attraction. GQDs were released and the fluorescence
restorated. According to the linear relationship between the fluorescence
intensity ratio I/I0 (I0 and I were the fluorescence intensities of the sensing
system in the absence and presence of heparin, respectively) and the concen-
trations of heparin, quantitative analysis of method heparin can be estab-
lished. Based on the same test principle, chondroitin sulfate can also be
determined rapidly and sensitively.

4. Conclusion
GQDs’ exceptional optical and electronic properties make them powerful
tools in the construction of sensing system, such as optical sensor and elec-
trochemical sensor. At the same time, their optoelectronics properties make
them excellent nanomaterials for a very wide variety of applications.64 PEC
sensors and ECL sensors based on GQDs have been developed gradually.
We have discussed that these nanoparticles have great potential in the
development of analytical sensors and biosensors. However, there are many
problems that have not been resolved during the synthesis of GQDs, such as
high-quality GQDs, appropriate control of size distribution uniformity, and
few ways of optical and electric properties tuning, and so on. Moreover, some
sensing mechanisms of sensors and biosensors constructed on the basis of
GQDs, their derivates or combination with other materials are not fully
understood.
In the fields of medical and pharmaceutical science, GQDs have been
used in the detection of biomolecule (e.g. DNA, antigen and enzyme, etc.)
and drug for clinical diagnosis and molecular screening of drug. We expected
the application is not only for sensing and GQDs would have more develop-
ment by virtue of advantages comparing with other QDs. They should be
further applied in studying the mechanism of drug effect, therapeutical effect,
pathogenesis and so on.
Although optical sensor based on GQDs developed rapidly, electrochemi-
cal sensor still needed to be improved. In addition, optoelectronics conver-
sion capacity is very important for GQDs research, so more studies in
optoelectronics conversion properties would be needed to extend their appli-
cation in nanomedicine fields. There is still much work to explore and
research the application of GQDs thoroughly.

b2227_P2-V1_Ch-04.indd 71 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

72  X. Zhang, J. Wang & G. Yang

References
1. Shen J, Zhu Y, Yang X, Li C. Graphene quantum dots: emergent nanolights for bioimag-
ing, sensors, catalysis and photovoltaic devices. Chem Comm 2012; 48: 3686–3699.
2. Zhang Z, Zhang J, Chen N, Qu L. Graphene quantum dots: An emerging material for
energy-related applications and beyond. Energy Environ Sci. 2012; 5: 8869–8890.
3. Li L, Wu G, Yang G, Peng J, Zhao J, Zhu J. Focusing on luminescent graphene quantum
dots: Current status and future perspectives. Nanoscale 2013; 5: 4015–4039.
4. Ting SL, Ee SJ, Ananthanarayanan A, Leong KC, Chen P. Graphene quantum dots func-
tionalized gold nanoparticles for sensitive electrochemical detection of heavy metal ions.
Electrochim Acta 2015; 172: 7–11.
5. Huang H, Liao L, Xu X, Zou M, Liu F, Li N. The electron-transfer based interaction
between transition metal ions and photoluminescent graphene quantum dots (GQDs):
A platform for metal ion sensing. Talanta 2013; 117: 152–157.
6. Fan Z, Li Y, Li X, Fan L, Zhou S, Fang D, Yang S. Surrounding media sensitive photo-
luminescence of boron-doped graphene quantum dots for highly fluorescent dyed crys-
tals, chemical sensing and bioimaging. Carbon 2014; 70: 149–156.
7. Amjadi M, Shokri R, Hallaj T. A new turn-off fluorescence probe based on graphene
quantum dots for detection of Au (III) ion. Spectrochim Acta 2016; 153: 619–624.
8. Dong Y, Li G, Zhou N, Wang R, Chi Y, Chen G. Graphene quantum dot as a green and
facile sensor for free chlorine in drinking water. Anal Chem 2012; 84: 8378–8382.
9. Wang L, Zheng J, Yang S, Wu C, Liu C, Xiao Y, Li Y, Qing Z, Yang R. Two-photon sensing
and imaging of endogenous biological cyanide in plant tissues using graphene quantum
dot/gold nanoparticle conjugate. Appl Mater Interfaces 2015; 7: 19509–19515.
10. Chen Y, Dong Y, Wu H, Chen C, Chi Y, Chen G. Electrochemiluminescence sensor for
hexavalent chromium based on the graphene quantum dots/peroxodisulfate system.
Electrochim Acta 2015; 151: 552–557.
11. Punrat E, Maksuk C, Chuanuwatanakul S, Wonsawat W, Chailapakul O. Polyaniline/
graphene quantum dot-modified screen-printed carbon electrode for the rapid determina-
tion of Cr (VI) using stopped-flow analysis coupled with voltammetric technique. Talanta
2016; 150: 198–205.
12. Xu L, Mao W, Huang J, Li S, Huang K, Li M, Xia J, Chen Q. Economical, green route
to highly fluorescence intensity carbon materials based on ligninsulfonate/graphene
quantum dots composites: Application as excellent fluorescent sensing platform for detec-
tion of Fe3+ ions. Sensor ctuat B-Chem 2016; 230: 54–60.
13. Zhang C, Cui Y, Song L, Liu X, Hu Z. Microwave assisted one-pot synthesis of graphene
quantum dots as highly sensitive fluorescent probes for detection of iron ions and pH
value. Talanta 2016; 150: 54–60.
14. Sun H, Gao N, Wu L, Ren J, Wei W, Qu X. Highly photoluminescent amino-­
functionalized graphene quantum dots used for sensing copper ions. Chem Eur J 2013;
19: 13362–13368.
15. Liu M, Liu T, Li Y, Xu H, Zheng B, Wang D, Du J, Xiao D. A FRET chemsensor based
on graphene quantum dots for detecting and intracellular imaging of Hg2+. Talanta 2015;
143: 442–449.
16. Chakraborti H, Sinha S, Ghosh S, Pal SK. Interfacing water soluble nanomaterials with
fluorescence chemosensing: Graphene quantum dot to detect Hg2+ in 100% aqueous
­solution. Mater Lett 2013; 97: 78–80.

b2227_P2-V1_Ch-04.indd 72 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Application of GQDs in Medical and Pharmaceutical Analyses 73

17. Shi B, Zhang L, Lan C, Zhao J, Su Y, Zhao S. One-pot green synthesis of oxygen-
rich nitrogen-doped grapheme quantum dots and their potential application in pH-sensi-
tive photoluminescence and detection of mercury (II) ions. Talanta 2015; 142:
131–139.
18. Hua M, Wang C, Qian J, Wang K, Yang Z, Liu Q, Mao H, Wang K. Preparation of gra-
phene quantum dots based core-satellite hybrid spheres and their use as the ratiometric
fluorescence probe for visual determination of mercury (II) ions. Anal Chim Acta 2015;
888: 173–181.
19. Qi Y, Zhang M, Fu Q, Liu R, Shi G. Highly sensitive and selective fluorescent detection
of cerebral lead (II) based on graphene quantum dot conjugates. Chem Commun 2013;
49: 10599–10601.
20. Park M, Ha HD, Kim YT, Jung JH, Kim SH, Kim DH, Seo TS. Combination of a sample
pretreatment microfluidic device with a photoluminescent graphene oxide quantum dot
sensor for trace lead detection. Anal Chem 2015; 87: 10969–10975.
21. Qian Z, Shan X, Chai L, Chen J, Feng H. A fluorescent nanosensor based on graphene
quantum dots–aptamer probe and graphene oxide platform for detection of lead (II) ion.
Biosens Bioelectron 2015; 68: 225–231.
22. Liu Z, Xiao J, Wu X, Lina L, Weng S, Chen M, Cai X, Lin X. Switch-on florescent strategy
based on N and S co-doped graphene quantum dots (N-S/GQDs) for monitoring
pyrophosphate ions in synovial fluid of arthritis patients. Sensor Actuat B-Chem 2016;
229: 217–224
23. Lu J, Yan M, Ge L, Ge S, Wang S, Yan J, Yu J. Electrochemiluminescence of blue-­
luminescent graphene quantum dots and its application in ultrasensitive aptasensor for
adenosine triphosphate detection. Biosensor Bioelectron 2013; 47: 271–277.
24. Liu J, Zhang X, Cong Z, Chen Z, Yang H, Chen G. Glutathione-functionalized graphene
quantum dots as selective fluorescent probes for phosphate-containing metabolites.
Nanoscale 2013; 5: 1810–1815.
25. Wu Z, Li W, Chen J, Yu C. A graphene quantum dot-based method for the highly sensitive
and selective fluorescence turn on detection of biothiols. Talanta 2014; 119: 538–543.
26. Liu Z, Gong Y, Fan Z. Cysteine detection using a high-fluorescence sensor based on a
nitrogen-doped graphene quantum dot–mercury (II) system. J Lumin 2016; 175:
129–134.
27. Lin L, Song X, Chen Y, Rong M, Wang Y, Zhao L, Zhao T, Chen X. Europium-decorated
graphene quantum dots as a fluorescent probe for label-free, rapid and sensitive detection
of Cu2+ and L-cysteine. Anal Chim Acta 2015; 891: 261–268.
28. Zhang Q, Song C, Zhao T, Fu H, Wang H, Wang Y, Kong D. Photoluminescent sensing
for acidic amino acids based on the disruption of graphene quantum dots/europium ions
aggregates. Biosensor Bioelectron, 2015; 65: 204–210.
29. Achadu OJ, Uddin I, Nyokong T. The interaction between graphene quantum dots
grafted with polyethyleneimine and Au@Ag nanoparticles: Application as a fluorescence
“turn-on” nanoprobe. J Photoch Photo A 2016; 324: 96–105.
30. Jua J, Zhang R, Chen W. Photochemical deposition of surface-clean silver nanoparticles
on nitrogen-doped graphene quantum dots for sensitive colorimetric detection of
­glutathione. Sensor Actuat B-Chemical 2016; 228: 66–73.
31. Wang L, Tricard S, Yue P, Zhao J, Fang J, Shen W. Polypyrrole and graphene quantum
dots @ Prussian Blue hybrid film on graphite felt electrodes: Application for amperometric
determination of L-cysteine. Biosensor Bioelectron 2016; 77: 1112–1118.

b2227_P2-V1_Ch-04.indd 73 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

74  X. Zhang, J. Wang & G. Yang

32. Ou J, Zhu Y, Kong Y, Ma J. Graphene quantum dots/β-cyclodextrin nanocomposites:

A novel electrochemical chiral interface for tryptophan isomer recognition. Electrochem
Commun 2015; 60: 60–63.
33. Zhang T, Zhao H, Fan G, Li Y, Li L, Quan X. Electrolytic exfoliation synthesis of
boron doped graphene quantum dots: A new luminescent material for electrochemilumi-
nescence detection of oncogene microRNA-20a. Electrochim Acta 2016; 190:
1150–1158.
34. Hu T, Zhang L, Wen W, Zhang X, Wang S. Enzyme catalytic amplification of miRNA-155
detection with graphene quantum dot-based electrochemical biosensor. Biosensor
Bioelectron 2016; 77: 451–456.
35. Zhao J, Chen G, Zhu L, Li L. Graphene quantum dots-based platform for the fabrication
of electrochemical biosensors. Electrochem Commun 2011; 13: 31–33.
36. Qian Z, Shan X, Chai L, Ma J, Chen J, Feng H. DNA nanosensor based on biocompatible
graphene quantum dots and carbon nanotubes. Biosensor Bioelectron 2014; 60: 64–70.
37. Qian Z, Shan X, Chai L, Chen J, Feng H. Simultaneous detection of multiple DNA
­targets by integrating dual-color graphene quantum dot nanoprobes and carbon nano-
tubes. Chem Eur 2014; 20: 16065–16069.
38. Wang G, Fang X, Wu X, Hu X, Li Z. Label-free and ratiometric detection of nuclei acids
based on graphene quantum dots Utilizing cascade amplification by nicking endonuclease
and catalytic G-quadruplex DNAzyme. Biosensor Bioelectron 2016; 81: 214–220.
39. Bhatnagar D, Kumar V, Kumar A. Inderpreet Kaur. Graphene quantum dots FRET based
sensor for early detection of heart attack in human. Biosensor Bioelectron 2016; 79:
495–499.
40. Zou F, Zhou H, Tan TV, Kim J, Koh K, Lee J. Dual-Mode SERS-fluorescence immuno-
assay using graphene quantum dot labeling on one-dimensional aligned magnetoplas-
monic nanoparticles. Appl Mater Interfaces 2015; 7: 12168–12175.
41. Pei H, Zhu S, Yang M, Kong R, Zheng Y, Qu F. Graphene oxide quantum dots@silver
core-shell nanocrystals as turn-on fluorescent nanoprobe for ultrasensitive detection of
prostate specific antigen. Biosensor Bioelectron 2015; 74: 909–914.
42. Zhang L, Li L, Ma C, Ge S, Yan M, Bian C. Detection of α-fetoprotein with an ultrasensi-
tive electrochemiluminescence paper device based on green-luminescent nitrogen-doped
graphene quantum dots. Sensors and Actuators B: Chemical 2015; 221: 799–806.
43. Li L, Li W, Ma C, Yang H, Ge S, Yu J. Paper-based electrochemiluminescence immuno-
device for carcinoembryonic antigen using nanoporous gold-chitosan hybrids and gra-
phene quantum dots functionalized Au@Pt. Sensors Actuators B: Chem 2014; 202:
314–322.
44. Poon CY, Li Q, Zhang J, Li Z, Dong C, Lee AWM, Chan WH, Li HW. FRET-based
modified graphene quantum dots for direct trypsin quantification in urine. Analytica
Chimica Acta 2016; 917: 64–70
45. Li X, Zhu S, Xu B, Ma K, Zhang J, Yang B, Tian W. Self-assembled graphene quantum
dots induced by cytochrome c: A novel biosensor for trypsin with remarkable fluorescence
enhancement. Nanoscale 2013; 5: 7776–7779.
46. Weng S, Liang D, Qiu H, Liu Z, Lin Z, Zheng Z, Liu A, Chen W, Lin X. A unique turn-
off fluorescent strategy for sensing dopamine based on formed polydopamine (pDA)
using graphene quantum dots (GQDs) as florescent probe. Sensors Actuators B: Chem
2015; 221: 7–14.

b2227_P2-V1_Ch-04.indd 74 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Application of GQDs in Medical and Pharmaceutical Analyses 75

47. Zhao J, Zhao L, Lan C, Zhao S. Graphene quantum dots as effective probes for label-free fluo-
rescence detection of dopamine. Sensors Actuators B: Chem 2016; 223: 246–251.
48. Zhou X, Ma P, Wang A, Yu C, Qian T, Wu S, Shen J. Dopamine fluorescent sensors based
on polypyrrole/grapheme quantum dots core/shell hybrids. Biosensors Bioelectron 2015;
64: 404–410.
49. Li Y, Jiang Y, Mo T, Zhou H, Li Y, Li S. Highly selective dopamine sensor based on
graphene quantum dots self-assembled monolayers modified electrode. J Electroanal
Chem 2016; 767: 84–90.
50. Zhang Y, Wu C, Zhou X, Wu X, Yang Y, Wu H, Guo S, Zhang J. Graphene quantum
dots/gold electrode and its application in living cell H2O2 detection. Nanoscale 2013; 5:
1816–1819.
51. Wu X, Tian F, Wang W, Chen J, Wu M, Zhao JX. Fabrication of highly fluorescent
­graphene quantum dots using L-glutamic acid for in vitro/in vivo imaging and sensing.
J Mater Chem C 2013; 1: 4676–4684.
52. Zheng A, Cong Z, Wang J, Li J, Yang H, Chen G. Highly-efficient peroxidase-like catalytic
activity of grapheme dots for biosensing. Biosensor Bioelectron 2013; 49: 519–524.
53. Lin L, Song X, Chen Y, Rong M, Zhao T, Wang Y, Jiang Y, Chen X. Intrinsic peroxidase-
like catalytic activity of nitrogen-doped grapheme quantum dots and their application in
the colorimetric detection of H2O2 and glucose. Anal Chim Acta 2015; 869: 89–95.
54. Nirala NR, Abraham S, Kumar V, Bansal A, Srivastava A, Saxena PS. Colorimetric detec-
tion of cholesterol based on highly efficient peroxidase mimetic activity of graphene
quantum dots. Sensor Actuat B: Chem 2015; 218: 42–50.
55. Amjadi M, Manzoori JL, Hallaj T. Chemiluminescence of graphene quantum dots and its
application to the determination of uric acid. J Lumin 2014; 153: 73–78.
56. Wang Y, Zhang L, Liang R, Bai J, Qiu J. Using graphene quantum dots as photolumines-
cent probes for protein kinase sensing. Analy Chem 2013; 85: 9148–9155.
57. Liang R, Qiu W, Zhao H, Xiang C, Qiu J. Electrochemiluminescence resonance energy
transfer between graphene quantum dots and graphene oxide for sensitive protein kinase
activity and inhibitor sensing. Anal Chim Acta 2016; 904: 58–64.
58. Liu j, He X, Wang K, He D, Wang Y, Mao Y, Shi H, Wen L. A highly sensitive electro-
chemiluminescence assay for protein kinase based on double-quenching of graphene
quantum dots by G-quadruplex–hemin and gold nanoparticles. Biosensor Bioelectron
2015; 70: 54–60.
59. He T, Qi L, Zhang J, Huang Y, Zhang Z. Enhanced graphene quantum dot fluorescence
nanosensor for highly sensitive acetylcholinesterase assay and inhibitor screening. Sensor
Actuat B: Chem 2015; 215: 24–29.
60. Liu Y, Yan K, Okoth OK, Zhang J. A label-free photoelectrochemical aptasensor based on
nitrogen-doped graphene quantum dots for chloramphenicol determination. Biosensor
Bioelectron 2015; 74: 1016–1021.
61. Zhao H, Chang Y, Liu M, Gao S, Yu H, Quan X. A universal immunosensing strategy
based on regulation of the interaction between graphene and graphene quantum dots.
Chem Comm 2013; 49: 234–236.
62. Patra S, Roy E, Choudhary R, Tiwari A, Madhuri R, Sharma PK. Graphene quantum dots
decorated CdS doped graphene oxide sheets in dual action mode: As initiator and plat-
form for designing of nimesulide imprinted polymer. Biosens Bioelectron 2015; 89:
627–635.

b2227_P2-V1_Ch-04.indd 75 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

76  X. Zhang, J. Wang & G. Yang

63. Li Y, Sun H, Shi F, Cai N, Lu L, Su X. Multi-positively charged dendrimeric nanoparticles

induced fluorescence quenching of graphene quantum dots for heparin and chondroitin
sulfate detection. Biosens Bioelectron 2015; 74: 284–290.
64. Benítez-Martínez S, Valcárcel M. Graphene quantum dots in analytical science. Trends
Anal Chem 2015; 93–113.

b2227_P2-V1_Ch-04.indd 76 23-Jun-17 2:56:03 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Chapter 5

Graphene Quantum Dots for Food Analysis

Yongkang Ye* and Xiaodong Cao

School of Food Science and Engineering

Hefei University of Technology
193 Tunxi Road, Hefei 230009, P.R. China
*[email protected]

Due to quantum size confinement and edge effects, graphene quantum dots (GQDs)
have presented extraordinary properties, attracting extensive attention from scientists
in the field. This chapter focuses in the recent progress made in GQDs nanotechnology
as applied to food analysis. Typical examples are presented for detected objectives
(e.g. microorganisms, pesticides, toxins, or heavy ions) and different analysis methods
(optical, electrochemical, and chromatographic) in food analysis.

1. The Nature of Food Analysis

Food analysis is the discipline dealing with a series specific of analytical pro-
cedures or protocols for characterizing a wide variety of properties of foods,
including their composition, physicochemical properties, structure, sensory
attributes and so on. Food analysis also plays an important role in quality
assurance procedures in food processing, as well as in developing, evaluating
and identifying new food products or processes. It is relatively complex
because food analysis integrates and applies principles of multidisciplines such
as biology, chemistry, physics, mathematics, statistics, nutrition, and engi-
neering to identify and characterize ingredients in ordinary or new food
products, monitor the food processing techniques used, and guarantee the
quality, safety and nutritional value of the food supply.1 Undoubtedly, food

77

b2227_P2-V1_Ch-05.indd 77 23-Jun-17 2:56:26 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

78  Y. Ye & X. Cao

analysis is continuously requesting the reliable and up-to-date development

of more sensitive, efficient, and cost-effective methodologies to ensure the
safety, quality and traceability of foods.2 The old food analysis methods used
in the last two centuries based on the chemical analysis (wet-chemistry or
well-established classical methods) have evolved into the current powerful
instrumental techniques used in food laboratories.1,3 Nowadays, modern
instrumental analysis has led to significant improvements in fast response,
accuracy, dynamic range, high selectivity, precision, time- and/or labor-saving
with higher sample throughput, which greatly expands their practical applica-
tion range of food research and industry.
Analytical methods used in food analysis could be classified according
to their working principle (see Figure 1). There are usually several different
analytical techniques available to determine a particular property of a food

Figure 1. Methods of instrumental analysis applied in food domain.

b2227_P2-V1_Ch-05.indd 78 23-Jun-17 2:56:27 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Food Analysis 79

ingredient. Choosing the most appropriate technique for specific application

depends on the property of food or food process to be measured, the type of
food to be analyzed, and the reason for carrying out the analysis, which
requires a good knowledge of various techniques. Information about the vari-
ous analytical procedures available can be obtained from a number of differ-
ent sources. Every technique provides specific information or demands
proper preparation on the sample or components under study based on a
specific physical–chemical interaction, and all have their own advantages and
drawbacks when applied to food analysis. Even though an analytical and bio-
analytical procedure (sometimes including “gold-standard”) is routinely used
in the food laboratory or industry, because of the need to resulting fast, reli-
able and precise information on the quality and security, it is always being
pushed to seeking for more sensitive and selective technique for food analysis
as an alternative one. Despite the recent achievements in food analysis, there
still exist many challenges to be solved, such as how to detect untargeted
compounds and determine their identity in foods; fast detection of food-
borne pathogens to overcome labor/time-consuming problems in food
industry and laboratories; in vivo non-destructive detection; and very few
real-world applications of commercialized nanosensors or so devices for
in situ food detection, as well as the implementation of Green Analytical
Chemistry in food laboratories.2,4
Nanotechnology and nanomaterials have a significant influence on many
aspects of food industry, from the development of novel food products
(nanoadditives nanocapsules nanosprays), conservation or packaging nanoma-
terials (enabling controlled release of preservatives to extend the shelf life of
foods) to food safety and security devices (detection of food-borne pathogens,
toxins, chemical residues, adulteration, etc.) for fast analysis.2 Naturally, expo-
sure to nanomaterials in the human food chain has a potential risk which needs
additional toxicology studies on different types of nanomaterials to understand
how they can affect food safety. Blasco and Picó reviewed some current appli-
cations of nanomaterials in food (including food additives and contact materi-
als) and analytical approaches such as sample preparation, imaging techniques
and detection/characterization methods for determining and evaluating nano-
materials in foods.5 Most important aspect of nanomaterials in this context,
such as metal nanoparticles or nanoclusters, metal oxide nanoparticles, quan-
tum dots (QDs), carbon nanotubes, graphene, and various nanocomposites
amplify signals to achieve high sensitivities and introduce transduction princi-
ples such as enhanced fluorescence, chemiluminescence, Raman signals, elec-
trochemical signals, and higher catalytic activity to the biosensing platform. In
fact, these nanomaterials also play a crucial role in the miniaturization of

b2227_P2-V1_Ch-05.indd 79 23-Jun-17 2:56:27 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

80  Y. Ye & X. Cao

platforms with more comprehensive automation and multifunction or in the

development of new nanomaterial-based biosensing systems for more selective
and sensitive food analysis applications, although such analytical nanotechnol-
ogy applied to food industry is still an emerging field.4,6,7 These advantages
have inspired research in coupling nanomaterial-based biosensing techniques
with biomolecules, which have been applied in different areas such as analysis
for foods general,4,6 bacterial foodborne pathogens,7,8 contaminants,8,9 food
ingredients (i.e. carbohydrate),10 heavy metal ions,11 etc.

2. Graphene Quantum Dots (GQDs) in Food Analysis

As quite newly emerging nanoprobes, QDs with small particle size, quantum
confinement discrete effect, band structure, surface effect and quantum tun-
nel effect derived some physical and chemical properties with excellent optical
and electrical properties, which have been used in an expanded range from
material sciences, chemistry, biology and medicine to food field, stimulating
the development of detection techniques in food analysis.6,7,9–15 Among these
QD materials, low-cytotoxic and biocompatible carbon-based nanomaterials
have recently emerged as the most attractive candidates for promising bioana-
lytical tools, such as carbon nanodots or carbon dots (CDs) have used in
bioimaging applications,16 as well as constructing a sensing platform for
detection of food additives,17 toxins,18 microorganisms,19,20 DNA recogni-
tion,21 drug and chemical residue,22 food ingredients,23 food contaminates,24
pesticides,25 abuse additives.26,27
Similar to CDs, GQDs9,28 and nitrogen-doped graphene quantum dots
(N-GQDs)29 belong to one of the most fascinating graphene material families
and newly developed luminescent or electrochemical nanomaterials have been
concerned because of their superiority in chemical inertness, low toxicity,
good solubility, satisfactory surface grating and high biocompatibility. Because
of the structure of original graphene, GQDs have some special properties.

(1) Optical properties. GQDs typically show strong optical absorption in

the UV region with a long-tail extending into the visible range. Some
reasonable photoluminescence (PL) mechanisms have been classified as
follows: the quantum-confinement effect or conjugated π-domains by
the carbon core, the surface state by hybridization of the carbon back-
bone with different chemical groups, the molecule state by the fluores-
cent molecules connected on the surface or interior of the GQDs, and
the crosslink-enhanced emission (CEE) effect30; meanwhile some of

b2227_P2-V1_Ch-05.indd 80 23-Jun-17 2:56:27 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Food Analysis 81

GQDs reportedly showed electrochemical luminescence (ECL) behav-

iors with the well-defined ECL peak.31
(2) Cytotoxicity. The low cytotoxicity of GQDs has been verified by various
research groups, more cytotoxicological data and in vivo experiments
test exhibit that the biological safety of GQDs have great potential for
in vitro and in vivo bioimaging applications.31–33
(3) Electronic properties. GQDs were shown to be both excellent electron
donors and acceptors due to the nature of graphene structure. A large
number of theoretical works suggested that the formation of local
moments was at GQD edges and other defects.31 GQDs have been used
to modified electrodes to design and prepare electrochemical sensors
with high sensitivity and selectivity.34,35

2.1. Microorganisms and food-borne pathogens

Microbiologists have developed reliable culture-based techniques for food-
borne pathogen detection, which are considered to be the “gold-standard”
over decades; however, they remain as complicated and time consuming pro-
cedures. The introduction of genetic-based technologies makes feasible
developing high-sensitive and specific screening assays for the fast and simple
detection of microorganisms and food-borne pathogens. Shi et al. developed
a fluorescence resonance energy transfer (FRET) biosensor based on GQDs
paired with gold nanoparticles (AuNPs) for Staphylococcus aureus specific
gene sequence recognition. The limit of detection (LOD) of this FRET
­biosensor was around 1 nM for S. aureus gene detection.36

2.2. Pesticides
Pesticides are compounds collection of insecticides, fungicides, herbicides,
molluscicides, rodenticides, plant growth regulators and repellents,8 which
contribute significantly to the contamination of the food chain. Due to the
high water solubility of most pesticides, their action mechanism, their high
level of toxicity and the widespread use in agriculture, there is a critical need
for highly sensitive and selective analytical methods for residue analysis of
these pollutants. Zor et al. reported on the synthesis of a multifunctional
composite material based on GODs, magnetic silica beads, and molecularly
imprinted polypyrrole, which enables a rapid, simple and sensitive platform
for pesticide tributyltin (as a model) detection, even in complex mediums
such as seawater without any sample treatment.37

b2227_P2-V1_Ch-05.indd 81 23-Jun-17 2:56:27 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

82  Y. Ye & X. Cao

2.3. Toxins
Toxins cover a large variety of molecules and can be grouped as plant/bacte-
rial toxins, mycotoxins and marine toxins. The consequences of consuming
food contaminated with toxins range from mild gastrointestinal problems to
death depending on the type of toxin and its concentration. Mycotoxins are
secondary metabolites naturally produced by fungal species that grow on
nuts, fruits and grains, both in the field, after harvest and during storage.
Their presence in food supply is due to direct contamination of plants or their
products or by contamination of animal feeds.4 Toxin detection methods fea-
tured with short analytical time, low cost, high sensitivity and easy adaptability
for in situ measurement are most wanted. Wang et al. developed a two-photo
excitation nanosensor using GQDs/AuNPs conjugate for sensing and imag-
ing endogenous biological CN−. With the benefit of the high quenching
efficiency of AuNPs and excellent two-photon properties of GQDs, the sens-
ing system can achieve a low detection limit and deeper penetration depth
without interference from background signals of a complex biological envi-
ronment, thus realizing sensing and imaging of CN− in different types of plant
tissues and even monitoring CN− removal in food processing.38

2.4. Heavy metal ions and chemical residues

As heavy metal ions and chemical residues severely harm human health, it is
important to develop simple, sensitive and accurate methods for their detection
in food industry. Yan et al. developed a sensitive detection of Hg2+ method
based on quenching effect of Hg2+ on N-GQDs with a relatively low detection
limit of 0.032 mM.29 Mohammad-Rezaei et al. prepared Fe3O4/GQDs mag-
netic nanocomposite for the preconcentration and determination of bisphenol
A (BPA) in drinking water samples using high-performance l­iquid chromatog-
raphy with a wide dynamic linear range (from 0.1 to 300 ng mL-1), and low
detection limit (12.3 pg mL-1) by using the ultraviolet detector.39 Huang et al.
proposed a sensing strategy for BPA was designed based on GQDs and peroxi-
dase, the quenching PL intensity of the GQDs was proportional to the con-
centration of BPA with a detection limit of 0.4 nM.40 Zhang et al. used GQDs
as the stationary phase for gas chromatographic capillary column separation of
alkanes and aromatic isomers.41 He et al. developed a simple and sensitive PL
method for the hydroquinone detection by using GQDs which simultaneously
serve as a peroxidase-mimicking catalyst and a PL indicator.42 Jian et al. attached
GQDs to the surface of a glassy carbon electrode (GCE) via electrostatic self-
assembly strategy, which exhibited high electrochemical activity and good
selectivity for the oxidation of hydroquinone and catechol.35 Ni’s group

b2227_P2-V1_Ch-05.indd 82 23-Jun-17 2:56:27 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Food Analysis 83

developed a resonance light scattering method for the analysis of phenol in

different types of industrial water.43 Jiang et al. modified gold electrode by
GQDs and Ag nanocubes (AgNCs) can be used as a novel and sensitive
enzyme-free H2O2 sensor.44

2.5. Veterinary drug residues

Determination of veterinary drug residues has become a routine procedure
and maximum residue limits have been established for drug residues in quality
control and safety of food. Zhao et al. used GQDs to fabricate an electro-
chemical sensor for the determination of acetaminophen for quality control
in the pharmaceutical industry as well as in food analysis.34

2.6. Food adulteration

Non-authentic or fraud food products of the adulteration spread all over the
world. Defrauders replace original substance partially or completely with
more easily available and cheap substance to seek greater interests.45
Determination of food authenticity and identification of adulteration have
become an important question in quality control and safety of food supply.
Due to a melamine-tainted milk scandal in 2008, Zhu’s group proposed a
rapid and sensitive fluorescence sensing system for melamine based on charge
transfer quenching of the fluorescence of GQDs in the presence of Hg2+.46

3. Choice of Food Analysis Methods

3.1. Optical detection
(1) Bioimaging methods
Wang et al. developed applications in chemical sensors and biological imagings
using two-photon excitation with near-infrared (NIR) photons as the excitation
source.38 The GQDs were assembled on the surface of peptide-functionalized
AuNPs through π−π stacking of GQDs and peptide to form the AuNP–PEP–
GQDs nanosensor. Before CN− addition, AuNPs displayed extremely high
quenching efficiency to GQDs due to the overlap between the emission spec-
trum of as-prepared GQDs and the absorption spectrum of AuNPs. In the
presence of CN−, AuNPs could be etched effectively and the disassembly of
nanosensor can thus induce fluorescence recovery of GQDs Figure 2(A). After
the response of the CN− sensor was achieved in vitro, two-photon excitation
confocal fluorescence imaging was performed to demonstrate the applicability
of endogenous CN− imaging in plant cells and tissues Figure 2(B).

b2227_P2-V1_Ch-05.indd 83 23-Jun-17 2:56:27 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

84  Y. Ye & X. Cao

Figure 2. (A) Schematic illustration of AuNP–PEP–GQDs nanosensor for CN− assay; (B)
Two-photon excitation images of (A) the fresh cassava cell, (B) cassava cell pretreated by soak-
ing and washing, and (C) 200 mM CN− preincubated cassava cell after incubation with AuNP–
PEP–GQDs (2 nM) for 2 h. Scale bar: 10 mm. Reused with permission from Ref. [38].
American Chemical Society.

(2) Fluorescence methods

Most GQD applications in food analysis are concentrated in the field of
­fluorescence, while practical mechanism of the PL of fluorescent GQDs is
still an open debate. Ni’s group used GQDs as fluorophores to detect the

b2227_P2-V1_Ch-05.indd 84 23-Jun-17 2:56:28 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Food Analysis 85

800 mIn 800 1

–4
0.24 × 10 M
1 800

FL Intensity
0 600 0.8 1.04

Fluorescence Intensity
1
400
600 .
2
.. 200
0.6
9.24 k = 0.936
600 0.4
FL Intensity

0 b = –0.0155
0 2 4 6 8 10 12
0.2 R = 0.9989
12 Time (min) –4
LOD = 8.4 × 10 M
400 0
400 2 4 6 8 10
4
Concentration (×10 M)

200 200

0 0
400 450 500 550 600 400 450 500 550 600
Wavelength (nm) Wavelength (nm)
(A) (B)
Figure 3. (A) Fluorescence response of GQDs in the presence of 9.6 mM hydroquinone,
6.0 mg L-1 HRP and 1.04 × 10-4 M H2O2 in pH 7.4 PBS buffer at different times.
Inset: (1) time-dependent fluorescence changes of GQDs with hydroquinone at 452 nm;
(2) fluorescence and GQDs in the absence and presence of hydroquinone; (B) The fluores-
cence quenching effect of hydroquinone at different concentrations in the presence of
HRP and H2O2 (reaction time: 8 min). Inset: fluorescence quenching effect vs. concentra-
tion of added hydroquinone. Reused with permission from Ref. [47]. The Royal Society
of Chemistry.

trace amount of hydroquinone in water samples. The quenching of

the fluorescence intensity of the GQDs in the presence of the quinone
intermediates is a measure of the discrimination of hydroquinone from
catechol, resorcinol and other compounds.47 When the hydroquinone–
HRP–H2O2 solution was added to a GQD solution and excited at 362 nm,
the observed, initially strong fluorescence was significantly quenched as
a function of increasing reaction time and concentration of added
­hydroquinone (Figure 3).
Ye’s team reported a one-pot method to prepare water-soluble N-GQDs
by using citric acid and glycine as carbon and nitrogen sources, respectively.29
And then as-prepared N-GQDs (1–4 nm) exhibit blue fluorescence with a
higher quantum yield (vs. GQDs). Due to the coordination occurring
between Hg2+ and functional groups on the surface of N-GQDs, the fluores-
cence of N-GQDs was quenched efficiently (Figure 4), the fluorescence
“turn-off” label-free sensor for Hg2+ was also achieved with the addition of
biothiols (cysteine or glutathione).
Chen and his co-worker also reported a colorimetric sensor for the detec-
tion of glutathione-based on the intrinsic peroxidase-like activity of Ag nano-
particles (AgNPs) supported on the N-GQDs.48 Figure 5 illustrated a
mechanism of the detection for glutathione. By the addition of biothiols
(glutathione) into the oxTMB (tetramethyl benzidine) solution, oxTMB can

b2227_P2-V1_Ch-05.indd 85 23-Jun-17 2:56:28 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

86  Y. Ye & X. Cao

Figure 4. The schematic illustration of the mechanism of the N-GQD-based detection of

Hg2+ and biothiols. Reused with permission from Ref. [29]. The Royal Society of Chemistry.

Figure 5. Schematic illustration of the mechanism for colorimetric detection of glutathione-

based on AgNPs N-GQDs TMB H2O2 system. Reused with permission from Ref. [48].
Elsevier B.V.

be reduced into TMB, resulting in the decrease of absorption peak at 652

nm, which is proportional to the concentration of added glutathione.
Huang et al. found that the fluorescence of GQDs could be quenched
with bisphenol A (BPA) in the presence of a H2O2 and horseradish peroxidase
(HRP) catalysis system (illustrated in Figure 6).40 The proposed method has
the opportunity to become a candidate for the sensing of BPA in real food
packaging samples.

b2227_P2-V1_Ch-05.indd 86 23-Jun-17 2:56:29 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Food Analysis 87

Figure 6. The schematic illustration of the BPA detection principle. Reused with permission
from Ref. [40]. The Royal Society of Chemistry.

(3) Fluorescence resonance energy transfer (FRET) methods

Bhatnagar et al. reported a simple, rapid and sensitive FRET-based immu-
nosensor for detection of an antibody using amine-functionalized GQDs.49
The fluorescence activity of the nanoprobe was further investigated with Gr
that results in quenching of the fluorescence intensity of so-prepared GQDs
nanoprobe. Finally, quantification of target was achieved by adding antigen
on antibody-modified GQDs/Gr, expelling stacked Gr apart from GQDs
nanoprobe and resulting in fluorescence recovery (sensing mechanism of
FRET is shown in Figure 7).
Shi et al. developed a FRET biosensor based on GQDs and AuNPs pairs
for Staphylococcus aureus specific gene sequence detection.36 The sensing
mechanism of FRET biosensor was realized by immobilization of capture
probes on GQDs and conjugation of reporter probes on AuNPs, and then
target oligos co-hybridized with capture probes and reporter probes to form
a sandwich structure which brought GQDs and AuNPs to close proximity to
trigger FRET effect (Figure 8). Then quenching effect decreased with the
decrease of target oligos with a detection limit of 1 nM.

(4) Electrochemiluminescent (ECL) methods

Dai’s group proposed a strategy for highly sensitive ECL detection of DNA
which was based on site-specific cleavage of endonuclease combined with the
excellent ECL activity of GQDs and bidentate chelation of the dithiocarba-
mate DNA probe assembly (Figure 9).50 This signal-off ECL DNA biosensor

b2227_P2-V1_Ch-05.indd 87 23-Jun-17 2:56:29 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

88  Y. Ye & X. Cao

Figure 7. (A) Bioconjugation of monoclonal antibody (anti-cTnI) with amine functionalized

GQDs using EDC–NHS chemistry. (B) Schematic mechanism of immunosensing based on
specific interaction of antibody modified GQDs with graphene. Reused with permission from
Ref. [49]. Elsevier B.V.

Figure 8. The sensing mechanism of the proposed GQDs–AuNPs FRET biosensor for
S. aureus gene detection. Reused with permission from Ref. [36]. Elsevier B.V.

was an alternative method for the highly sensitive determination of DNA.

Because different probes could specifically bind to different targets, this plat-
form could be conveniently combined with other specific biological recogni-
tion events for extensive biosensing application in food domine.50

b2227_P2-V1_Ch-05.indd 88 23-Jun-17 2:56:30 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Food Analysis 89

Figure 9. Schematic illustration of the ECL biosensor based on GQDs combined with endo-
nuclease cleavage and bidentate chelation. Reused with permission from Ref. [50]. American
Chemical Society.

3.2. Electrochemical detection

Zhao et al. used GQDs to modify the GCE to obtain a simple electrochemical
sensor.34 The obtained sensor could successfully overcome overlapping oxida-
tion peaks to realize simultaneous detection of acetaminophen and ascorbic
acid. Differential pulse voltammetry (DPV) was used to detect acetaminophen
and ascorbic acid on the GQD-modified GCE (Figure 10). The dynamic
range for the simultaneous determination of acetaminophen and ascorbic acid
was 1–200 mmol L-1 and 25–1400 mmol L-1, respectively.
Jian et al. used an electrostatic self-assembly strategy to attach the GQDs
to the surface of a GCE which was applied to investigate the electrochemical
behavior of hydroquinone (HQ) and catechol (CC) (Figure 11). The GQDs-
modified electrode exhibited high-electrochemical activity, good selectivity
and high sensitivity for the simultaneous determination of HQ and CC.35

3.3. Chromatographic detection

Razmi’s team described the preparation, characterization and application of a
magnetic nanocomposite of Fe3O4/GQDs as an adsorbent for magnetic solid

b2227_P2-V1_Ch-05.indd 89 23-Jun-17 2:56:30 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

90  Y. Ye & X. Cao

Figure 10. (A) DPVs of the GQD-modified GCE in PBS w/o ascorbic acid and acetami-
nophen; (B) CVs of the GQD-modified GCE in PBS (pH = 6.5) with ascorbic acid and aceta-
minophen at various scan rates. Reused with permission from Ref. [34]. The Royal Society of
Chemistry.

phase extraction (MSPE).39 As discussed earlier, the method was used to

determine the trace amount of BPA in mineral water samples, which were
stored in polyethylene bottles, and to investigate the leakage of BPA from
polyethylene bottles to the mineral water after direct exposure to sunlight
(Figure 12).39
Zhang et al. reported GQDs as the stationary phase for gas chromato-
graphic capillary column separation of alkanes and aromatic isomers with

b2227_P2-V1_Ch-05.indd 90 23-Jun-17 2:56:31 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Food Analysis 91

Figure 11. Schematic drawing of the synthesis of GQDs from pyrolysis citric acid and elec-
trochemical oxidize HQ and CC on GQDs/GCE. Reused with permission from Ref. [35].
Elsevier Ltd.

Figure 12. Chromatograms of (A) fresh mineral water; (B) mineral water kept in plastic bot-
tles and directly exposed to sunlight for one week; (C) mineral water kept in plastic bottles and
directly exposed to sunlight for one week and spiked with 20 ng mL-1 BPA. Reused with
permission from Ref. [39]. The Royal Society of Chemistry.

3-aminopropyldiethoxymethyl silane as the coupling reagent.41 GQD

coated capillary columns have a high efficiency of ethylbenzene, styrene,
xylene, propylbenzene, alkanes, and dichlorobenzene isomers separation at
low temperature. The thermodynamics of the separation were investigated
with the separation of xylene, propylbenzene, dichlorobenzene, styrene
and EB isomers at difference temperatures from 40 to 90ºC. The results

b2227_P2-V1_Ch-05.indd 91 23-Jun-17 2:56:31 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

92  Y. Ye & X. Cao

Figure 13. Effect of temperature on the chromatograms on the GQD coated capillary
­column (22 m long × 0.32 mm i.d.) for the separation of: (A) ethylbenzene and styrene;
(B) ethylbenzene, p-Xylene and o-Xylene; (C) propylbenzene isomers; (D) dichlorobenzene
isomers. Reused with Permission from Ref. [41]. The Royal Society of Chemistry.

showed the retention time of analytes on the GQD-coated capillary

column gradually decreased with the increases in column temperature
­
(Figure 13).

References
1. Picó Y. Chemical Analysis of Foods: Techniques and Applications. 1st Edn. Academic Press
is an imprint of Elsevier: 225 Wyman Street, Waltham, MA 02451, USA 2012.
2. Cifuentes A. Food analysis: Present, future, and foodomics. ISRN Anal Chem 2012;
2012: Article ID 801607.
3. Nielsen SS. Food Analysis. In Food Analysis, 4th Edn., Nielsen SS (Ed), Springer:
New York, 2010: p. 6.

b2227_P2-V1_Ch-05.indd 92 23-Jun-17 2:56:32 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Food Analysis 93

4. Valdés MG, Valdés González AC, García Calzón JA, Díaz-García ME. Analytical nano-
technology for food analysis. Microchim Acta 2009; 166: 1–19.
5. Blasco C, Picó Y. Determining nanomaterials in food. TrAC Trend Anal Chem 2011; 30:
84–99.
6. Pérez-López B, Merkoçi A. Nanomaterials based biosensors for food analysis applications.
Trends Food Sci Tech 2011; 22: 625–639.
7. Stephen Inbaraj B, Chen BH. Nanomaterial-based sensors for detection of foodborne
bacterial pathogens and toxins as well as pork adulteration in meat products. J Food Drug
Anal 2016; 24: 15–28.
8. McGrath TF, Elliott CT, Fodey TL. Biosensors for the analysis of microbiological and
chemical contaminants in food. Anal Bioanal Chem 2012; 403: 75–92.
9. Sharma R, Ragavan KV, Thakur MS, Raghavarao KS. Recent advances in nanoparticle
based aptasensors for food contaminants. Biosens Bioelectron 2015; 74: 612–627.
10. Wang Y, Qu K, Tang L, Li Z, Moore E, Zeng X, Liu Y, Li J. Nanomaterials in carbohy-
drate biosensors. TrAC Trends Anal Chem 2014; 58: 54–70.
11. Cui L, Wu J, Ju H. Electrochemical sensing of heavy metal ions with inorganic, organic
and bio-materials. Biosens Bioelectron 2015; 63: 276–286.
12. Eltzov E, Guttel S, Low Yuen Kei A, Sinawang PD, Ionescu RE, Marks RS. Lateral flow
immunoassays — from paper strip to smartphone technology. Electroanalysis 2015; 27:
2116–2130.
13. Huang X, Aguilar ZP, Xu H, Lai W, Xiong Y. Membrane-based lateral flow immunochro-
matographic strip with nanoparticles as reporters for detection: A review. Biosens
Bioelectron 2016; 75: 166–180.
14. Syed MA. Advances in nanodiagnostic techniques for microbial agents. Biosens Bioelectron
2014; 51: 391–400.
15. Wang X, Niessner R, Tang D, Knopp D. Nanoparticle-based immunosensors and immu-
noassays for aflatoxins. Anal Chim Acta 2016; 912: 10–23.
16. Jiang C, Wu H, Song X, Ma X, Wang J, Tan M. Presence of photoluminescent carbon
dots in Nescafe(R) original instant coffee: Applications to bioimaging. Talanta 2014;
127: 68–74.
17. Gao Z, Wang L, Su R, Huang R, Qi W, He Z. A carbon dot-based “off-on” fluorescent
probe for highly selective and sensitive detection of phytic acid. Biosens Bioelectron 2015;
70: 232–238.
18. Wang B, Chen Y, Wu Y, Weng B, Liu Y, Lu Z, Li CM, Yu C. Aptamer induced assembly
of fluorescent nitrogen-doped carbon dots on gold nanoparticles for sensitive detection of
AFB1. Biosens Bioelectron 2016; 78: 23–30.
19. Lai IP-J, Harroun SG, Chen S-Y, Unnikrishnan B, Li Y-J, Huang C-C. Solid-state syn-
thesis of self-functional carbon quantum dots for detection of bacteria and tumor cells.
Sensor Actuat B Chem 2016; 228: 465–470.
20. Duan N, Gong W, Wang Z, Wu S. An aptasensor based on fluorescence resonance energy
transfer for multiplexed pathogenic bacteria determination. Anal Methods 2016; 8:
1390–1395.
21. Loo AH, Sofer Z, Bousa D, Ulbrich P, Bonanni A, Pumera M. Carboxylic carbon quan-
tum dots as a fluorescent sensing platform for DNA detection. ACS Appl Mater Interfaces
2016; 8: 1951–1957.

b2227_P2-V1_Ch-05.indd 93 23-Jun-17 2:56:32 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

94  Y. Ye & X. Cao

22. Xue M, Zhang L, Zhan Z, Zou M, Huang Y, Zhao S. Sulfur and nitrogen binary doped
carbon dots derived from ammonium thiocyanate for selective probing doxycycline in liv-
ing cells and multicolor cell imaging. Talanta 2016; 150: 324–330.
23. Liu J, Chen Y, Wang W, Feng J, Liang M, Ma S, Chen X. “Switch-on” fluorescent sensing
of ascorbic acid in food samples based on carbon quantum dots-MnO2 Probe. J Agr Food
Chem 2016; 64: 371–380.
24. Liu G, Chen Z, Jiang X, Feng D, Zhao J, Fan D, Wang W. In-situ hydrothermal synthesis
of molecularly imprinted polymers coated carbon dots for fluorescent detection of bisphe-
nol A. Sensor Actuat B-chem 2016; 228: 302–307.
25. Hou J, Dong G, Tian Z, Lu J, Wang Q, Ai S, Wang M. A sensitive fluorescent sensor for
selective determination of dichlorvos based on the recovered fluorescence of carbon dots-
Cu(II) system. Food Chem 2016; 202: 81–87.
26. Yue X, Zhu W, Ma S, Yu S, Zhang Y, Wang J, Wang Y, Zhang D, Wang J. Highly sensitive
and selective determination of tertiary butylhydroquinone in edible oils by competitive
reaction induced “on–off–on” fluorescent switch. J Agr Food Chem 2016; 64: 706–713.
27. Xu H, Yang X, Li G, Zhao C, Liao X. Green synthesis of fluorescent carbon dots for selec-
tive detection of tartrazine in food samples. J Agr Food Chem 2015; 63: 6707–6714.
28. Wang Z, Yu J, Gui R, Jin H, Xia Y. Carbon nanomaterials-based electrochemical aptasen-
sors. Biosens Bioelectron 2016; 79: 136–149.
29. Yan Z, Qu X, Niu Q, Tian C, Fan C, Ye B. A green synthesis of highly fluorescent nitro-
gen-doped graphene quantum dots for the highly sensitive and selective detection of
mercury(II) ions and biothiols. Anal Methods 2016; 8: 1565–1571.
30. Zhu S, Song Y, Zhao X, Shao J, Zhang J, Yang B. The photoluminescence mechanism in
carbon dots (graphene quantum dots, carbon nanodots, and polymer dots): Current state
and future perspective. Nano Res 2015; 8: 355–381.
31. Zhang Z, Zhang J, Chen N, Qu L. Graphene quantum dots: An emerging material for
energy-related applications and beyond. Energy Environ Sci 2012; 5: 8869–8890.
32. Shen J, Zhu Y, Yang X, Li C. Graphene quantum dots: Emergent nanolights for bioimag-
ing, sensors, catalysis and photovoltaic devices. Chem Commun 2012; 48: 3686–3699.
33. Guo X, Mei N. Assessment of the toxic potential of graphene family nanomaterials. J Food
Drug Anal 2014; 22: 105–115.
34. Zhao C, Liu Z, Xu W, Chen M, Weng S, Xu L, Cai Q. A glassy carbon electrode based
on graphene quantum dots (GQDs) for simultaneous detection of acetaminophen and
ascorbic acid. Anal Methods 2015; 7: 8877–8881.
35. Jian X, Liu X, Yang H, Guo M, Song X, Dai H, Liang Z. Graphene quantum dots modi-
fied glassy carbon electrode via electrostatic self-assembly strategy and its application.
Electrochim Acta 2016; 190: 455–462.
36. Shi J, Chan C, Pang Y, Ye W, Tian F, Lyu J, Zhang Y, Yang M. A fluorescence resonance
energy transfer (FRET) biosensor based on graphene quantum dots (GQDs) and gold
nanoparticles (AuNPs) for the detection of mecA gene sequence of Staphylococcus aureus.
Biosens Bioelectron 2015; 67: 595–600.
37. Zor E, Morales-Narvaez E, Zamora-Galvez A, Bingol H, Ersoz M, Merkoci A. Graphene
quantum dots-based photoluminescent sensor: A Multifunctional composite for pesticide
detection. ACS Appl Mater Interfaces 2015; 7: 20272–20279.
38. Wang L, Zheng J, Yang S, Wu C, Liu C, Xiao Y, Li Y, Qing Z, Yang R. Two-photon
sensing and imaging of endogenous biological cyanide in plant tissues using graphene

b2227_P2-V1_Ch-05.indd 94 23-Jun-17 2:56:32 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Food Analysis 95

quantum dot/gold nanoparticle conjugate. ACS Appl Mater Interfaces 2015; 7:

19509–19515.
39. Mohammad-Rezaei R, Razmi H, Abdollahi V, Matin AA. Preparation and characteriza-
tion of Fe3O4/graphene quantum dots nanocomposite as an efficient adsorbent in mag-
netic solid phase extraction: Application to determination of bisphenol A in water samples.
Anal Meth 2014; 6: 8413–8419.
40. Huang H, Feng Z, Li Y, Liu Z, Zhang L, Ma Y, Tong J. Highly sensitive detection of
bisphenol A in food packaging based on graphene quantum dots and peroxidase. Anal
Methods 2015; 7: 2928–2935.
41. Zhang X, Ji H, Zhang X, Wang Z, Xiao D. Capillary column coated with graphene quan-
tum dots for gas chromatographic separation of alkanes and aromatic isomers. Anal
Methods 2015; 7: 3229–3237.
42. He Y, Sun J, Feng D, Chen H, Gao F, Wang L. Graphene quantum dots: Highly active
bifunctional nanoprobes for nonenzymatic photoluminescence detection of hydroqui-
none. Biosens Bioelectron 2015; 74: 418–422.
43. Sun R, Wang Y, Ni Y, Kokot S. Graphene quantum dots and the resonance light scattering
technique for trace analysis of phenol in different water samples. Talanta 2014; 125:
341–346.
44. Jiang Y, Li Y, Li Y, Li S. A sensitive enzyme-free hydrogen peroxide sensor based on a
chitosan–graphene quantum dot/silver nanocube nanocomposite modified electrode.
Anal Meth 2016; 8: 2448–2455.
45. Kamal M, Karoui R. Analytical methods coupled with chemometric tools for determining
the authenticity and detecting the adulteration of dairy products: A review. Trends Food
Sci Tech 2015; 46: 27–48.
46. Li L, Wu G, Hong T, Yin Z, Sun D, Abdel-Halim ES, Zhu JJ. Graphene quantum dots
as fluorescence probes for turn-off sensing of melamine in the presence of Hg2+. ACS Appl
Mater Interfaces 2014; 6: 2858–2864.
47. Li Z, Sun R, Ni Y, Kokot S. A novel fluorescent probe involving a graphene quantum
dot–enzyme hybrid system for the analysis of hydroquinone in the presence of toxic
­resorcinol and catechol. Anal Meth 2014; 6: 7420–7425.
48. Ju J, Zhang R, Chen W. Photochemical deposition of surface-clean silver nanoparticles
on nitrogen-doped graphene quantum dots for sensitive colorimetric detection of
­glutathione. Sensor Actuat B-chem 2016; 228: 66–73.
49. Bhatnagar D, Kumar V, Kumar A, Kaur I. Graphene quantum dots FRET based sensor
for early detection of heart attack in human. Biosens Bioelectron 2016; 79: 495–499.
50. Lou J, Liu S, Tu W, Dai Z. Graphene quantums dots combined with endonuclease
cleavage and bidentate chelation for highly sensitive electrochemiluminescent DNA
­
­biosensing. Anal Chem 2015; 87: 1145–1151.

b2227_P2-V1_Ch-05.indd 95 23-Jun-17 2:56:32 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Chapter 6

Fluorescent Graphene Quantum Dots

for Bioimaging

Shuhua Li, Zetan Fan, Fanglong Yuan and Louzhen Fan*

Department of Chemistry, Beijing Normal University

Beijing 100875, P.R. China
*[email protected]

Owing to the outstanding characteristics of high aqueous solubility, low cytotoxicity,

good biocompatibility, stable photoluminescence (PL) as well as superior resistance
to photobleaching and blinking compared with traditional organic dyes and
semiconductor quantum dots (QDs), the emerging fluorescent graphene quantum
dots (GQDs) have attracted considerable attention for their potential applications
in the biomedical field as fluorescent imaging agents to track molecular targets in
cells or body. This allows us to obtain direct evidences for the understanding of the
underlying molecular mechanisms and dynamic processes of the biological system.
Previous chapters have given a detailed introduction on the synthesis, properties and
applications of GQDs in food detection and biosensing. In this chapter, we will place
emphasis on the recent advances and future prospects of GQDs in bioimaging both
in vitro and in vivo.

1. Introduction of Graphene Quantum Dots (GQDs)

in Fluorescence Imaging
Imaging techniques to visualize physiological or pathophysiological changes
in both cells and body are essential for the diagnosis and treatment of disease
as well as for research into the basic processes of life.1 A number of imaging
approaches such as radioisotope labeling, electron spin resonance spectros-
copy, computed tomography, single-photon emission computed tomography,

97

b2227_P2-V1_Ch-06.indd 97 23-Jun-17 2:56:53 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

98  S. Li et al.

magnetic resonance imaging, positron emission tomography and ultrasound

have been widely applied in the field of bioimaging. 2–7 Despite this plethora
of visualization techniques, fluorescence imaging is emerging as an important
alternative because of its high sensitivity, less-invasive, operational simplicity,
safety, and cost-effectiveness.8
Fluorescence imaging can be divided into two broad categories: in vitro
fluorescence imaging (mainly cellular imaging) and in vivo fluorescence imag-
ing. Cellular imaging has long relied on light microscopy and is attracting
increasing attention as technological advances provide enhanced capabilities.
Versatile fluorescence-based techniques, particularly confocal microscopy and
wide-field microscopy, can be used to quantify cellular phenotypic changes
and investigate the role of a particular target in in vitro disease studies.
Moreover, the advent of fluorescence microscopy and other sophisticated
techniques has enabled routine studies of dynamic processes in living cells.
These fluorescence techniques can deliver the necessary resolution to image
certain cellular organelles and track proteins in the nucleus, endoplasmic
reticulum and Golgi apparatus, and other biomolecules in living cells, 9 so that
valuable information about the dynamics of intracellular networks, signal
transduction, and intercellular interactions can be obtained. On the other
hand, in vivo fluorescence imaging resembles fluorescence microscopy in that
both use a low-light camera and appropriate filters to collect fluorescence
emission light from samples, but differs in that it works at a macroscopic
level.10 The objects of imaging are whole-body small animals instead of cells
in culture dishes or on slides. This extension into the in vivo setting allows
visualization of biology in its intact and native physiological state.
Fluorescence imaging depends on the inherent property of the imaging
agents, which mainly include fluorescent proteins, organic dyes, semiconduc-
tor QDs and fluorescent carbon nanomaterials. The discovery of the green
fluorescence protein (GFP) has fundamentally changed the landscape of bio-
medical research in the past decades. However, it has a less defined role in
clinical applications due to the low emission wavelength of 510 nm which
overlaps with the auto-fluorescence of tissues.11–13 Organic dyes are the most
commonly used fluorescence imaging agents, but their poor chemical stability
and photostability make long-term bioimaging difficult.14,15 Semiconductor
QDs have been regarded as the promising alternative owing to their high
brightness and photostability.16,17 The fluorescence emission of semiconduc-
tor QDs can be tuned across a wide range from UV-blue (ZnS)18 to near-
infrared (NIR) (CdS/HgS/CdS, InP, InAs)19,20 through the visible (CdE,
with E = S, Se, Te).21 However, typical contents of heavy metal elements such
as cadmium or lead in semiconductor QDs have generated much concern

b2227_P2-V1_Ch-06.indd 98 23-Jun-17 2:56:53 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Fluorescent Graphene Quantum Dots for Bioimaging 99

with respect to in vivo or even in vitro uses. And the blinking characteristics
of semiconductor QDs make single-molecule tracking difficult. In addition,
because of a much larger size than a biomolecule, semiconductor QDs might
affect the dynamics and functions of the target molecules and create artificial
clusters while associating with multiple targets. It is worth mentioning that
GQDs, as a new emerging star in the field of fluorescent carbon nanomateri-
als, are also exciting in this regard.22,23 GQDs are advantageous compared to
fluorescent proteins, organic dyes, and semiconductor QDs due to their
chemical inertness, low cytotoxicity, good biocompatibility as well as superior
resistance to photobleaching and blinking.24,25 In particular, functional
groups such as carboxyl, amino, carbonyl, hydroxyl, and other groups on the
surface of GQDs result in their high hydrophilicity and readiness for further
functionalization with various organic, polymeric, or biological species,26
which expand the range and heighten the sensitivity and selectivity of bioim-
aging. By taking advantage of the unique properties of GQDs coupled with
other merits such as low cost and ease of synthesis, a large number of research
groups have investigated the potential applications of GQDs in cellular imag-
ing.27,28 Moreover, GQDs with up-conversion fluorescence or red fluores-
cence have been successfully synthesized by designing appropriate carbon
precursors or synthetic strategies,29,30 which further expands the applications
of GQDs in in vivo imaging. Following will focus on the recent advances of
GQDs for in vitro and in vivo imaging, respectively, and discuss issues and
prospects in such applications.

2. GQDs for In Vitro Imaging

2.1. Cancer cellular imaging
The possibility of using GQDs as fluorescent labels for cancer cellular imaging
was first demonstrated by Zhu et al. in 2011.31 They prepared fluorescent
GQDs via one-step solvothermal method from graphene oxide (GO) and
­performed GQDs uptake and bioimaging experiments on human osteosarcoma
(MG-63) cells by the confocal fluorescence microscope (Figures 1(A)–1(C)).
After incubating MG-63 cells with GQDs at 37oC in a CO2 incubator for 12
h, green and green-yellow fluorescence emissions at the excitation wavelength
of 405 and 488 nm were clearly observed in the cell cytoplasm, indicating the
translocation of GQDs through the cell membrane. The low cytotoxicity of
GQDs as also demonstrated by cell viability tests using the methylthiazolyldi-
phenyl-tetrazolium bromide (MTT) assay. As shown in Figure 1(D), addition
of up to 400 mg of GQDs to 150 mL of culture medium (104 cells) did not

b2227_P2-V1_Ch-06.indd 99 23-Jun-17 2:56:53 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

100  S. Li et al.

Figure 1. Cellular imaging and cellular toxicity of GQDs. (A)–(C) are washed cells imaged
under bright field, 405 nm and 488 nm excitations, respectively. (D) Effect of GQDs on
MG-63 cells viability. Reprinted with permission from Ref. [31]. Copyright 2011 Royal
Society of Chemistry.

weaken the cell activity significantly, suggesting that GQDs possess low toxic-
ity and could be used in bioimaging at high concentrations. Since then,
GQDs have been used to label a variety of cancer cells such as human cervical
carcinoma cells (HeLa),32–37 human lung cancer cells (A-549),38–41 murine
alveolar macrophage cells (MH-S),42 human hepatic cancer cells (Huh7),43
human breast cancer cells (MCF-7),44 and ovarian cancer cells (A2780).45 All
these studies suggested the low cytotoxicity and excellent ­biocompatibility of
GQDs, thus they could be used as an eco-friendly material for biolabeling
and bioimaging.
Although GQDs emitting blue to red fluorescence have shown potential
application in cancer cellular imaging, most researches only concentrated on

b2227_P2-V1_Ch-06.indd 100 23-Jun-17 2:56:53 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Fluorescent Graphene Quantum Dots for Bioimaging 101

the uptake of bare GQDs by cancer cells without considering the selectivity
and function of the agents. Targeted cancer cellular imaging, as a fundamen-
tal research, promotes cancer detection and targeted therapeutic modalities
for effective cancer chemotherapy with improved safety. GQDs are generally
composed of sp2- and sp3-hybridized carbon nanostructures in the form of
conjugated carbon clusters functionalized with abundant periphery oxygen-
containing functional groups, imparting them with suitability for subsequent
functionalization with organic, polymeric, inorganic or biological species by
p–p conjugation or chemical modifications.46–53 Therefore, targeted cancer
cellular imaging based on functionalized GQDs has also been developed.
Fluorescent GQDs were prepared and conjugated with folic acid (FA) to
demonstrate highly selective and specific cancer cellular imaging by Wang
et al.53 The FA-conjugated GQDs (GQDs-FA) were incubated with three cell
types that express FA receptor (FR) at different levels to demonstrate that FA
retained its binding affinity to FR after conjugation with GQDs. As shown in
Figure 2, the fluorescence of GQDs-FA in HeLa cells is considerably stronger
than that in A549 and HEK293A (human embryonic kidney cell line) cells,
which expressed FA receptor (FR) at a low level, indicating that GQDs-FA
were internalized via FR-induced endocytosis. The GQDs-FA were then
employed as carriers of the anticancer drug doxorubicin (DOX) for targeted
cell delivery.
The cell nucleus is critical to various important cellular events, including
metabolism, heredity and reproduction.54 Nucleus-staining is the first step to
revealing the nucleus morphology, to investigating the nuclear function, and
to transferring the agent to the cell nucleus.55 In most of the cellular imaging
experiments, GQDs, like other nanomaterials, were located only in the cyto-
plasm. However, single-layer GQDs prepared by refluxing Vulcan CX-72
carbon black with concentrated nitric acid were found to be able to enter the
cell nucleus (Figure 3).56 This was the first time for GQDs to label the cell
nucleus, but the researchers did not come up with a convincing explanation
of GQDs entering the nucleus. Immediately following these findings, Wang
et al. also demonstrated the nucleus labeling property of polyethylenimine
functionalized GQDs (GQDs-PEI) synthesized by a new convenient solvo-
thermal method which combined the “top-down” cutting, reducing and
functionalizing GO sheets in one-step.57 They deduced that the nucleus per-
meability of GQDs-PEI may be attributed to the ultrasmall size of GQDs-
PEI and high concentration of positively charged nitrogen atoms of PEI,
which also exhibits an excellent membrane-disruption ability. Cell nucleus
imaging using GQDs offers a further application in the diagnosis of diseased
phenotypes and the targeted therapy of cancer.

b2227_P2-V1_Ch-06.indd 101 23-Jun-17 2:56:53 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

102  S. Li et al.

Figure 2. Confocal laser scanning microscopy images of (A) HeLa, (B) A549 and (C)
HEK293A cells incubated with GQDs-FA (20.0 mg/mL) at 37oC for 10 min. Left column:
bright field images. Middle column: fluorescence image with Hoechst nuclear stained (blue).
Right column: GQDs fluorescence image (green). Reprinted with permission from Ref. [53].
Copyright 2014 Elsevier.

2.2. Stem cellular imaging

Stem cells are central to the understanding of the native development and
tissue regeneration, which give rise to tissue progenitor cells and in turn dif-
ferentiate into tissue-forming cells.58,59 When stem cells or their progeny are
applied to regenerate tissues and organs, there is a critical need to delineate
the relative contribution to the regenerated tissues and organs from delivered
cells versus host cell. Thus, long-term and cytocompatible labeling of stem
cells is critically needed in the field of regeneration medicine for understand-
ing their fate, migration and contribution to the regenerating tissues.

b2227_P2-V1_Ch-06.indd 102 23-Jun-17 2:56:54 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Fluorescent Graphene Quantum Dots for Bioimaging 103

Figure 3. Confocal laser scanning microscope images of MCF-7 cells labeled with GQDs.
(A) Fluorescent image, (B) bright-field image, (C) merged fluorescent and bright-field image,
and (D) section analysis. Reprinted with permission from Ref. [56]. Copyright 2012 Royal
Society of Chemistry.

Although GQDs can penetrate into the cancer cells effortlessly for imaging in
various studies, stem cells labeling still poses a considerable challenge.
Zhang et al. firstly used stabilizer-free GQDs as a fluorescent labeling
agent in long-term stem cellular imaging.60 Bright yellow fluorescent GQDs
with fluorescence quantum yield (QY) of 14% have demonstrated direct and
easy penetration into three different kinds of stem cells, neurospheres cells
(NSCs), pancreas progenitor cells (PPCs) and cardiac progenitor cells (CPCs)
without affecting their viability, proliferation or differentiation capacity. As
shown in Figure 4, the morphology of stem cells could be clearly discerned
after incubated with GQDs. They further investigated the uptake mechanism
and biocompatibilty of such GQDs with human neural stem cells (hNSCs).61
Transmission electron microscopy (TEM) images confirmed that the GQDs
were indeed internalized by the hNSCs via the endocytosis mechanism and

b2227_P2-V1_Ch-06.indd 103 23-Jun-17 2:56:54 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

104  S. Li et al.

Figure 4. Confocal fluorescence microscopy images of NSCs (A), PPCs (C) and CPCs (E)
with the yellow fluorescent GQDs incorporated at the excitation wavelength of 405 nm and
corresponding images under bright field (B, D, F). Reprinted with permission from Ref. [60].
Copyright 2012 Royal Society of Chemistry.

were located in the cytoplasm. And GQDs did not affect the self-renewal and
expression of the cell type-specific marker in hNSCs. Interestingly, the fluo-
rescent C60 nanoparticles which could easily penetrate into cancer cells were
not detected inside stem cells62 and CdS QDs were shown to be cytotoxic to

b2227_P2-V1_Ch-06.indd 104 23-Jun-17 2:56:55 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Fluorescent Graphene Quantum Dots for Bioimaging 105

stem cells.63 Therefore, such GQDs have a distinct advantage in terms of their
penetrating power to stem cells.
Cancer stem cells (CSCs), as a subpopulation of stem-like cells within
tumors, exhibit the characteristics of both stem cells and cancer cells.64 They
have the capacity to self-renew inside a tumor and to generate heterogeneous
lineages of cancer cells that drive cancer progression, metastasis and drug
resistance.65,66 CSCs have been proposed as the cause of tumor relapse and
are the relatively new target of cancer therapies, and it is of vital importance
and urgent necessity to successfully label CSCs. Guo et al. synthesized
­rhodamine B derivative-functionalized GQDs (RBD-GQDs) which can bind
to Fe3+ forming RBD-GQDs–Fe3+ with strong orange–red fluorescence of
43% QY.67 Such RBD-GQDs–Fe3+ were first demonstrated to be biomarkers
for pancreatic CSCs (Figure 5). Compared with other competing fluorescent

Figure 5. Confocal fluorescence microscopy images of pancreatic CSCs incubated with

(A) 10 mM, (B) 25 mM, and (C) 50 mM Fe3+ for 5 h, followed by further incubation with
RBD-GQDs (25 mg/L) for 0.5 h.67 Reprinted with permission from Ref. [67]. Copyright
2015 American Chemical Society.

b2227_P2-V1_Ch-06.indd 105 23-Jun-17 2:56:56 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

106  S. Li et al.

nanomaterials, these GQDs internalized in CSCs are expected to be more

practicable in drug delivery and cancer treatment.

2.3. Real-time molecular tracking in live cells

Real-time molecular tracking of dynamic cellular processes requires that fluo-
rescent tags are bright, photostable, biocompatible, targeted and of molecular
size to minimize physical hindrance. Zheng et al. demonstrated that oxygen-
ated GQDs (∼2 nm and ∼2 kDa) can be readily conjugated with any proteins
(including neuropeptide Y, bovine serum albumin, immunoglobulin G, con-
canavalin A, insulin, and nerve growth factor), without impairing the func-
tionalities of the protein or largely altering the size, weight, and charge
state.68 In this seminal work, insulin-conjugated GQDs were synthesized to
track the real-time dynamics (distribution, internalization, and recycling) of
insulin receptors in 3T3-L1 adipocytes (Figures 6(A)–6(E)) using total inter-
nal reflection fluorescence microscopy (TIRFM), which evanescently and
selectively illuminated the thin plasmalemmal region. This study discovered,
for the first time, that internalization and recycling of insulin receptors in

Figure 6. (A) TIRFM image of a 3T3-L1 adipocyte after 1 h incubation of insulin-GQDs.

Scale bar is 5 mm. (B) Fluorescent membrane patch of insulin-GQDs/insulin receptor (type I).
(C) Endocytosis of membrane patches into a vesicle (type II). (D) Exocytosis of a vesicle with
insulin-GQDs/insulin receptor (type III). (E) Transient approaching and retrieval of insulin-
GQDs/insulin receptor with vesicle (type IV). Scale bars is 0.2 mm. Reprinted with permission
from Ref. [68]. Copyright 2013 American Chemical Society.

b2227_P2-V1_Ch-06.indd 106 23-Jun-17 2:56:57 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Fluorescent Graphene Quantum Dots for Bioimaging 107

adipocytes are oppositely regulated by apelin and tumor necrosis factor-alpha

(TNFα), demonstrating the great potential of GQDs as universal and superior
fluorescent tags for real-time molecular imaging in live cells.

3. GQDs for In Vivo Imaging

In vivo imaging can delineate and measure, at the macroscopic level, biologi-
cal processes that are occurring at the cellular and molecular level.69
Fluorescence imaging using GQDs has already been developed for in vitro
applications in cellular biology, but is still at an early stage of development as
a whole-animal in vivo imaging technique. Due to the auto-fluorescence and
light scattering background of biological specimens in short-wavelength
region, GQDs with exceptional two-photon excitation (up-conversion) prop-
erty are desired for in vivo imaging. The quasi-zero-dimensional (0D) GQDs
with a single-atomic layer gives rise to several advantages for potential two-
photon fluorescence imaging (TPFI) in vivo. First, the bandgap and fluores-
cence of GQDs can be effectively tuned by doping heteroatoms to the
π-conjugated system.70–74 It can be expected that doping nitrogen through
introducing strong electron-donating groups such as dimethylamido could
result in a red shift of fluorescence and achieve longer emission and excitation
wavelengths, 75 which are less scattered by biotissues and more practical for in
vivo imaging. Second, GQDs without passivation by any surfactant can exhibit
strong fluorescence induced by a pronounced quantum confinement and edge
effect,76–78 which could also impart a larger two-photon absorption cross-sec-
tion and deeper penetration in turbid tissues.79–81 Third, the large rigid
π-conjugated electronic structure of GQDs can also improve the intramolecu-
lar charge transfer efficiency and therefore enhance the two-photon absorp-
tion to achieve larger imaging depth.82 Nitrogen-doped GQDs (N-GQDs)
with a high two-photon absorption cross-section of 48,000 Göppert–Mayer
(GM) units have been found particularly suitable for in vivo imaging.30
Alternatively, the availability of GQDs emitting in the red region is par-
ticularly important for in vivo imaging, since deep-red and NIR light exhibit
deeper tissue penetration (Figure 7(A)).83 It has been reported that the fluo-
rescence of GQDs can be tuned from deep ultraviolet to red by controlling
their size and shape, heteroatoms doping, and modifying their surfaces and
edges due to the remarkable quantum-confinement effect and edge effect.30
Most of the prepared GQDs were considered to be consisting of small sp2
clusters isolated within the sp3 carbon matrix,84–89 and the energy gap between
the p and p* states generally depends on the size of sp2 clusters or the conju-
gation length. Theoretical modelling and calculations have clearly shown that

b2227_P2-V1_Ch-06.indd 107 23-Jun-17 2:56:57 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

108  S. Li et al.

Figure 7. (A) The visualization of light penetration through the tissues according to its wave-
length. (B) Energy gap of π–π* transitions calculated based on DFT as a function of the num-
ber of fused aromatic rings. (C) Fluorescence spectra (c1) and high-resolution TEM image (c2)
of RF-GQDs.91 (A) Reprinted with permission from Ref. [83]. Copyright 2011 American
Cancer Society. (B) Reprinted with permission from Ref. [90]. Copyright 2010 John Wiley
and Sons. (C) Reprinted with permission from Ref. [91]. Copyright 2015 Royal Society of
Chemistry.

an sp2 cluster of more than 20 aromatic rings has an energy gap of around
2 eV (Figure 7(B)) which can convert into about 600 nm PL.90 Larger sp2
clusters have a lower energy gap and may emit at the red light region. Red
fluorescent GQDs (RF-GQDs) without any chemical modification were suc-
cessfully prepared via facile electrochemical exfoliation of graphite in K2S2O8
solution by Tan et al. (Figure 7(C)).91 The RF-GQDs were found to consist
of isolated sp2 domains with a diameter of about 3 nm, and the very active
SO4•- radicals produced from S2O82- acted as electrochemical ‘‘scissors’’ to
sharply cut the graphene sheets into small intact sp2 structures, known as the

b2227_P2-V1_Ch-06.indd 108 23-Jun-17 2:56:57 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Fluorescent Graphene Quantum Dots for Bioimaging 109

RF-GQDs we obtained. This is the first report on the direct observation of

‘‘molecular’’ sp2 domains with a diameter of about 3 nm that is directly
responsible for the red emission, but the QY of RF-GQDs is low, which limits
their application in in vivo imaging.
GQDs with excellent fluorescence properties and peroxidase-like activity
were prepared via one-step pyrolysis of L-glutamic acid and used for in vivo
imaging by Wu et al.42 The developed GQDs showed strong blue, green and
red fluorescence under the irradiation of ultra violet, blue and green light,
respectively. Moreover, the GQDs emitted NIR fluorescence in the range of
800–850 nm with the excitation-dependent manner. This NIR fluorescence
has a large Stokes shift of 455 nm, providing significant advantage for sensi-
tive imaging of biological targets. After injecting GQDs subcutaneously into
the back of nude mice and intramuscularly into the right back leg, the fluo-
rescence images of the mice under different excitation and emission filters
were obtained (Figure 8(A)). The detectable fluorescence region extended
with the longer excitation and emission wavelengths from the intramuscular
injection spot of the right back leg, demonstrating that longer wavelengths
for in vivo fluorescence imaging had better penetration ability than the short
ones. Afterwards, Wang’s research group prepared highly water-dispersible
GQDs with a deep-red emission peaking at 680 nm via a hydrothermal

Figure 8. (A) In vivo fluorescence imaging of mice injected GQDs subcutaneously (spot a)
and intramuscularly (spot b). The images were taken at various excitation wavelengths and
emission wavelengths indicated at the top of each image. Copyright 2013 Royal Society of
Chemistry. (B) Bright-field image (b1) and red-fluorescence image (b2) after subcutaneous
injection of GQDs in different areas. The excitation wavelength was 502–540 nm, and the
collected fluorescence channel was 695–775 nm. (A) Reprinted with permission from Ref.
[92]. (B) Reprinted with permission from Ref. [93]. Copyright 2014 Nature Publishing
Group.

b2227_P2-V1_Ch-06.indd 109 23-Jun-17 2:56:58 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

110  S. Li et al.

method with polythiophene derivatives (PT2) as the carbon source and

investigated the in vivo fluorescence imaging capability of such red fluorescent
GQDs.92 After the injection of GQDs aqueous solution into the back of a
nude mouse, the injection sites showed much higher fluorescence intensity
than the background signal produced by the mouse skin (Figure 8(B)).
Furthermore, no apparent fluorescence intensity decay was observed at the
injection sites, and the injected GQDs did not show evidence of obvious dif-
fusion even one week after injection. The preparation of GQDs with up-
conversion fluorescence or red fluorescence will further expand the applications
of GQDs in biomedicine field.

4. Summary and Outlook

This section discusses the recent developments of GQDs focusing on their
applications in cellular (including cancer cell and stem cell) imaging and in
vivo imaging. Despite the tremendous amount of exciting results reported in
the past few years in this field, there are still many challenges ahead towards
applications of GQDs-based imaging. First of all, the exploration of more
specific design and synthesis of high-quality GQDs with various sizes, shapes,
and surface functionalities for cell nucleus imaging and stem cellular imaging
at high resolution is still ongoing. Secondly, although the highest QY of
GQDs reported so far is 54.5% with blue fluorescence emission,42 the QY of
GQDs with red fluorescence emission is only 6%,93 much lower than tradi-
tional semiconductor QDs and organic dyes. Therefore, GQDs with high QY
in longer wavelength emission beyond the blue light region are highly desired
for effective tissue penetration and in vivo imaging. Finally, GQDs with a
higher two-photon absorption cross-section are expected for two-photon
fluorescence imaging in vivo, which exhibits inherent superiorities such as a
larger penetration depth, a minimized tissue auto-fluorescence background,
the avoidance of harmful UV or blue excitations and the reduction of photo-
damage in biotissues.93,94

References
1. Megason SG, Fraser SE. Cell 2007; 130: 784–795.
2. Mahmood U. IEEE Eng Med Biol Mag 2004; 23: 58–66.
3. Pomper MG, Hammoud DA. IEEE Eng Med Biol Mag 2004; 23: 28–37.
4. Hogemann D, Basilion JP. Eur J Nucl Med Mol Imaging 2002; 29: 400–408.
5. Hogemann D, Basilion JP, Weissleder R. Radiologe 2001; 41: 116–120.
6. Bremer C, Ntziachristos V, Mahmood U, Tung CH, Weissleder R. Radiologe 2001; 41:
131–137.

b2227_P2-V1_Ch-06.indd 110 23-Jun-17 2:56:58 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Fluorescent Graphene Quantum Dots for Bioimaging 111

7. Mahmood U, Weissleder R. Acad Radiol 2002; 9: 629–631.

8. Nagano T. Proc Jpn Acad Ser B 2010; 86: 837–847.
9. Marta FS, Ting AY. Nat Rev Mol Cell Biol 2008; 9: 929–943.
10. Rao JH, Anca DA, Yao HQ. Curr Opin Biotechnol 2007; 18: 17–25.
11. Hoffman RM. Methods Mol Med 2005; 111: 297–322.
12. Terakawa S. Tanpakushitsu Kakusan Koso 2004; 49: 1641–1646.
13. Tran PT, Paoletti A, Chang F. Methods 2004; 33: 220–225.
14. Chan WC, Maxwell DJ, Gao X, Bailey RE, Han M, Nie S. Curr Opin Biotechnol 2002;
13: 40–46.
15. Bruchez JM, Moronne M, Gin P, Weiss S, Alivisatos AP. Science 1998; 281:
2013–2016.
16. Baker M. Nat Meth 2010; 7: 957–962.
17. Michalet X, Pinaud FF, Bentolila A, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM,
Gambhir SS, Weiss S. Science 2005; 307: 538–544.
18. Weller H, Koch U, Gutierrez M, Henglein A. Ber Bunsenges Phys Chem 1984; 88:
649–656.
19. Eychmuller A, Mews A, Weller H. Chem Phys Lett 1993; 208: 59–62.
20. Guzelian AA, Katari JEB, Kadavanich AV, Banin U, Hamad K, Juban E, Alivisatos AP,
Wolters RH, Arnold CC, Heath JR. J Phys Chem 1996; 100: 7212–7219.
21. Mattoussi H, Mauro JM, Goldman ER, Anderson GP, Sundar VC,Mikulec FV, Bawendi
MG. J Am Chem Soc 2000; 122: 12142–12150.
22. Kusakabe K, Maruyama M. Phys Rev B: Condens Matter 2003; 67: 092406-1–4.
23. Son YW, Cohen ML, Louie SG. Nature 2006; 444: 347–349.
24. Li H, Kang Z, Liu Y, Lee S-T. J Mater Chem 2012; 22: 24230–24253.
25. Baker SN, Baker GA. Angew Chem Int Ed 2010; 49: 6726–6744.
26. Kong B, Zhu A, Ding C, Zhao X, Li B, Tian Y. Adv Mater 2012; 24: 5844–5848.
27. Wang ZF, Zeng HD, Sun LY. J Mater Chem C 2015; 3: 1157–1165.
28. Li LL, Wu GH, Yang GH, Peng J, Zhao JW, Zhu J-J. Nanoscale 2013; 5: 4015–4039.
29. Zhu S, Zhang J, Tang S, Qiao C, Wang L, Wang H, Liu X, Li B, Li Y, Yu W, Wang X,
Sun H, Yang B. Adv Funct Mater 2012; 22: 4732–4740.
30. Liu Q, Guo BD, Rao Z Y, Zhang BH, Gong JR. Nano Lett 2013; 13: 2436–2441.
31. Zhu S, Zhang J, Qiao C, Tang S, Li Y, Yuan W, Li B, Tian L, Liu F, Hu R, Gao H, Wei
H, Zhang H, Sun H, Yang B. Chem Commun 2011; 47: 6858–6860.
32. Pan DY, Guo L, Zhang JC, Xi C, Xue Q, Huang H, Li JH, Zhang ZW, Yu WJ, Chen ZW,
Li Z, Wu MH. J Mater Chem 2012; 22: 3314–3318.
33. Fan ZT, Li YC, Li XH, Fan LZ, Zhou SX, Fang DC, Yang SH. Carbon 2014; 70: 149–156.
34. Zhu X, Xiao X, Zuo X, Liang Y, Nan J. Part Part Syst Char 2014; 31: 801–809.
35. Hu C, Liu Y, Yang Y, Cui J, Huang Z, Wang Y, Yang L, Wang H, Xiao Y, Rong J. J Mater
Chem B 2013; 1: 39–42.
36. Chen S, Hai X, Xia C, Chen X-W, Wang J-H. Chem Eur J 2013; 19: 15918–15923.
37. Li N, Liang X, Wang L, Li Z, Li P, Zhu Y, Song J. J Nanopart Res 2012; 14:
1177–1185.
38. Xie WJ, Fu YY, Ma H, Zhang M, Fan LZ. Acta Chimi Sinica 2012; 70: 2169–2172.
39. Sun H, Wu L, Gao N, Ren J, Qu X. ACS Appl Mater Interfaces 2013; 5: 1174–1179.
40. Sun Y, Wang S, Li C, Luo P, Tao L, Wei Y, Shi G. Phys Chem Chem Phys 2013; 15:
9907–9913.

b2227_P2-V1_Ch-06.indd 111 23-Jun-17 2:56:58 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

112  S. Li et al.

41. Zhang X, Wang S, Liu M, Yang B, Feng L, Ji Y, Tao L, Wei Y. Phys Chem Chem Phys 2013;
15: 19013–19018.
42. Wu X, Tian F, Wang W, Chen J, Wu M, Zhao JX, J Mater Chem C 2013; 1:
4676–4684.
43. Kumar V, Singh V, Umrao S, Parashar V, Abraham S, Singh AK, Nath G, Saxena PS,
Srivastava A. RSC Adv 2014; 4: 21101–21107.
44. Zhou L, Geng J, Liu B. Part Part Syst Char 2013; 30: 1086–1092.
45. Zhang C, Liu Y, Xiong XQ, Peng LH, Gan L, Chen CF, Xu HB. Org Lett 2012; 14:
5912–5915.
46. Qiu J, Zhang R, Li J, Sang Y, Tang W, Gil PR, Liu H. Int J Nanomed 2015; 10:
6709–6724.
47. Jing Y, Zhu Y, Yang X, Shen J, Li C. Langmuir 2011; 27: 1175–1180.
48. Chen T, Yu H, Yang NW, Wang MD, Ding CD, Fu JJ. J Mater Chem B 2014; 2:
4979–4982.
49. Deng L, Liu L, Zhu C, Li D, Dong S. Chem Commun 2013; 49: 2503–2505.
50. Xue Q, Huang H, Wang L, Chen Z, Wu M, Li Z, Pan D. Nanoscale 2013; 5:
12098–12103.
51. Qian Z, Ma J, Shan X, Shao L, Zhou J, Chen J, Feng H. RSC Adv 2013; 3:
14571–14579.
52. Nigam P, Waghmode S, Louis M, Wangnoo S, Chavan P, Sarkar D. J Mater Chem B
2014; 2: 3190–3195.
53. Wang XJ, Sun X, Lao J, He H, Cheng TT, Wang MQ, Wang SJ, Huang F. Colloid Surface
B 2014; 122: 638–644.
54. Li C, Liu Y, Wu Y, Sun Y, Li F. Biomaterials 2013; 34: 1223–1234.
55. Horobin RW, Stockert JC, Doubell FR. Histochem Cell Biol 2013; 139: 623–637.
56. Dong YQ, Chen CQ, Zheng XT, Gao LL, Cui ZM, Yang HB, Guo CX, Chi YW, Li CM.
J Mater Chem 2012; 22: 8764–8766.
57. Wang H, Wang X. RSC Adv 2015; 5: 75380–75385.
58. Prockop DJ. Science 2001; 293: 211–212.
59. Parker GC, Kristeva MA, Eisenberg LM, Rao MS, Williams MA, Sanberg PR, English D.
Stem Cells Dev 2005; 14: 463–469.
60. Zhang M, Bai LL, Shang WH, Xie WJ, Ma H, Fu YY, Fang DC, Sun H, Fan LZ, Han M,
Liu CM, Yang SH. J Mater Chem 2012; 22: 7461–7467.
61. Shang WH, Zhang XY, Zhang M, Fan ZT, Sun Y, Han M, Fan LZ. Nanoscale 2014; 6:
5799–5806.
62. E YF, Bai LL, Fan LZ, Han M, Zhang XY, Yang SH. J Mater Chem 2011; 21: 819.
63. CZ Wang, E YF, Fan LZ, Wang ZH, Liu HB, Li YL, Yang SH, Li YL. Adv Mater 2007;
19: 3677–3681.
64. Yu Z, Pestell TG, Lisanti MP, Pestell RG. Int J Biochem Cell Biol 2012; 44: 2144–2151.
65. Bao Q, Zhao Y, Renner A, Niess H, Seeliger H, Jauch K-W, Bruns CJ. Cancers 2010; 2:
1629–1641.
66. Reya T, Morrison SJ, Clarke MF, Weissman IL. Nature 2001; 414: 105–111.
67. Guo RH, Zhou SX, Li YC, Li XH, Fan LZ, Voelcker NH. ACS Appl Mater Interfaces
2015; 7: 23958–23966.
68. Zheng XT, Than A, Ananthanaraya A, Kim D-H, Chen P. ACS Nano 2013; 7:
6278–6286.

b2227_P2-V1_Ch-06.indd 112 23-Jun-17 2:56:58 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Fluorescent Graphene Quantum Dots for Bioimaging 113

69. Biswal S, Resnick DL, Hoffman JM, Gambhir SS. Radiology 2007; 244: 651–671.
70. Li Y, Zhao Y, Cheng HH, Hu Y, Shi GQ, Dai LM, Qu LT. J Am Chem Soc 2012; 134:
15–18.
71. Guo BD, Liu Q, Chen ED, Zhu HW, Fang L, Gong JR. Nano Lett 2010; 10:
4975–4980.
72. Guo BD, Fang L, Zhang BH, Gong JR. Electron Lett 2011; 47: 663–664.
73. Guo BD, Fang L, Zhang BH, Gong JR. Insciences J 2011; 1: 80–89.
74. Li Q, Zhang S, Dai L, Li LS. J Am Chem Soc 2012; 134: 18932–18935.
75. Tetsuka H, Asahi R, Nagoya A, Okamoto K, Tajima I, Ohta R, Okamoto A. Adv Mater
2012; 24: 5333–5338.
76. Ponomarenko L, Schedin F, Katsnelson M, Yang R, Hill E, Novoselov K, Geim A. Science
2008; 320: 356–358.
77. Girit CO, Meyer JC, Erni R, Rossell MD, Kisielowski C, Yang L, Park CH, Crommie M,
Cohen ML, Louie SG. Science 2009; 323: 1705–1708.
78. Loh KP, Bao QL, Eda G, Chhowalla M. Nat Chem 2010; 2: 1015–1024.
79. Padilha, LA, Nootz G, Olszak PD, Webster S, Hagan DJ, Van Stryland EW, Levina L,
Sukhovatkin V, Brzozowski L, Sargent EH. Nano Lett 2011; 11: 1227–1231.
80. Luo ZT, Vora PM, Mele EJ, Johnson ATC, Kikkawa JM. Appl Phys Lett 2009; 94:
111909-1–3.
81. Trauzettel B, Bulaev DV, Loss D, Burkard G. Nat Phys 2007; 3: 192–196.
82. Collini E. Phys Chem Chem Phys 2012; 14: 3725–3736.
83. Agostinis P, Berg K, Cengel KA, Foster TH, Girotti AW, Gollnick SO, Hahn SM,
Hamblin MR, Juzeniene A, Kessel D, Korbelik M, Moan J, Mroz P, Nowis D, Piette J,
Wilson BC, Golab J. CA Cancer J Clin 2011; 61: 250–281.
84. Mattevi C, Eda G, Agnoli S, Miller S, Mkhoyan KA, Celik O, Mastrogiovanni D,
Granozzi G, Garfunkel E, Chhowalla M. Adv Funct Mater 2009; 19: 2577–2583.
85. Ishigami M, Chen JH, Cullen WG, Fuhrer MS, Williams ED. Nano Lett 2007; 7:
1643–1648.
86. Kudin KN, Ozbas B, Schniepp HC, Prud’homme RK, Aksay IA, Car R. Nano Lett 2007;
8: 36–41.
87. Gomez-Navarro C, Meyer JC, Sundaram RS, Chuvilin A, Kurasch S, Burghard M, Kern
K, Kaiser U. Nano Lett 2010; 10: 1144–1148.
88. Jung I, Field DA, Clark NJ, Zhu Y, Yang D, Piner RD, Stankovich S, Dikin DA, Geisler
H, Ventrice CA, Ruoff RS. J Phys Chem C 2009; 113: 18480–18486.
89. Wilson NR, Pandey PA, Beanland R, Young RJ, Kinloch IA, Gong L, Liu Z, Suenaga K,
Rourke JP, York SJ, Sloan J. ACS Nano 2009; 3: 2547–2556.
90. Eda G, Lin YY, Mattevi C, Yamaguchi H, Chen HA, Chen IS, Chen CW, Chhowalla M.
Adv Mater 2010; 22: 505–509.
91. Tan XY, Li YC, Li XH, Zhou SX, Fan LZ, Yang SH. Chem Commun 2015; 51:
2544–2546.
92. Ge J, Lan M, Zhou B, Liu W, Guo L, Wang H, Jia Q, Niu G, Huang X, Zhou H, Meng
X, Wang P, Lee CS, Zhang W, Han X. Nat Commun 2014; 5: 4596–4603.
93. Denk W, Strickler JH, Webb WW. Science 1990; 248: 73–76.
94. Helmchen F, Denk W. Nat Meth 2005; 2: 932–940.

b2227_P2-V1_Ch-06.indd 113 23-Jun-17 2:56:58 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Chapter 7

Graphene Quantum Dots for Drug Delivery

Yang Chen and Maoquan Chu*

Research Center for Translational Medicine at Shanghai East Hospital

150 Jimo Road, Shanghai 200120, P.R. China
School of Life Science and Technology, Tongji University
Shanghai 200092, P.R. China
*[email protected]

Graphene quantum dots (GQDs), sharing advantages and properties of both graphene
and semiconductor quantum dots (QDs) have been widely used for biomedical
application mainly in bioimaging and biosensing since they have small lateral size
and excellent elec­trochemical and optical properties, capability to bind with a variety
of aromatic biomolecules through a π–π stacking interaction and/or electrostatic
interaction, as well as fine water solubility, excellent biocompatibility and minimal
toxicity, which also made them the ideal materials for drug delivery. In this chapter,
we provide an overview of significant advances in GQDs-based carriers for delivery of
chemical and biological drugs.

1. Introduction
Nowadays, tremendous efforts have been contributed to the development of
targeted drug delivery systems. Among these systems, nanomaterials play
important roles, including improving the solubility of hydrophobic drugs,
achieving targeted delivery, controlling drug release and depressing toxic side
effects.1 An ideal nanomaterial for drug delivery needs to have excellent drug
loading capacity, active functional groups to conjugate drugs or targeting
antibodies, good biocompatibility, and low toxicity. Organic nanoparticles
like liposomes, micelles and inorganic nanoparticles like gold nanoparticles,
115

b2227_P2-V1_Ch-07.indd 115 23-Jun-17 2:57:21 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

116  Y. Chen & M. Chu

magnetic nanoparticles, silica nanoparticles and carbon-materials based drug

delivery systems are all attractive to researchers and a few of them have already
reached the final stages of development and even received approval from
Food and Drug Administration (FDA).2–5 However, the drug loading effi-
ciency of these nanocarriers should be further improved when used in vivo.1–5
Graphene quantum dots (GQDs), small fragments of graphene with small
lateral size range6 and large surface area can notably improve drug loading
because they can bind with a variety of biomolecules through a π–π stacking
interaction and/or electrostatic interaction. When functionalized to get active
atoms like oxygen and nitrogen during or after synthesis,7–9 they become more
easy to conjugate/bind with drugs and ligands.10–14 Meanwhile owing to
the inherent flexibility and small size of the GQDs, drug-GQDs conjugates
can penetrate the nucleus by diffusion and enhance the toxicity of drugs
to DNA.15 GQDs have special electrochemical and optical properties, their
intrinsic luminescence can help surveille the whole process of internalization,
intracel­lular distribution, and drug release. Upon irradiation, they can release
reactive singlet oxygen, indicating the applicability in chemo-photodynamic
therapy. Thus, GQDs make themselves a promising nanomaterial for drug
delivery and researchers have already made some achievements in this field. In
this chapter, recent advances in GQDs-based carriers for delivery of chemical
and biological drugs are described.

2. Transport Properties and Mechanism Across Cells of GQDs

If we can figure out the transport properties and cellular uptake mechanism
of GQDs, it will help us better understand the way GQDs deliver drugs .Wang
and her colleagues16 investigated the transport and cellular uptake properties
of GQDs across the Madin Darby Canine Kidney (MDCK) cell monolayer.
The experimental results revealed that GQDs crossed the cell membrane with
good permeability through a lipid raft-mediated transcytosis and the smaller
size GQDs exhibited higher membrane permeability. Shang et al.17 examined
the interactions of GQDs on human neural stem cells. Their results indicated
that GQDs were taken up through a concentration- and time-dependent
manner via the receptor-mediated endocytosis mechanism, suggesting that
GQDs might be biocompatible. Wang15 used MCF-7 cells as an example, and
found that energy-dependent endocytosis, clathrin and caveolae mediated
endocytosis were likely to happen in the cellular uptake of the GQD.
Therefore, GQDs may be excellent carriers for drug delivery since they
have good ability to penetrate across the cell membrane. And these multiple
endocytosis ways may contribute to the efficient cellular uptake of GQD

b2227_P2-V1_Ch-07.indd 116 23-Jun-17 2:57:21 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Drug Delivery 117

conjugates and increase the cytotoxicity of drugs to drug-resistant cancer

cells,18 since one of the generally accepted drug-resistant mechanisms is that
the active drug efflux pumps are overexpressed on the membrane of many
drug-resistant cells.19,20

3. GQDs-Based Carriers for Delivery of Chemical

and Biological Drugs
3.1. GQDs-based carriers for chemotherapy drug delivery
3.1.1. GQDs as drug carriers only
As mentioned above, GQDs can bind with a variety of aromatic biomolecules
through π–π stacking interaction and/or electrostatic interaction, therefore,
most of drugs are easy to be loaded. On the other hand, GQDs have good
membrane permeability and biocompatibility which can improve the
­pharmaceutical performance of the loaded drugs and are more effective to
drug-resistant cells. Some et al.21 reported that curcumin (Cur)-containing
GQDs had high anticancer activity both in vitro and in vivo. They prepared
three kinds of Cur-graphene composites: graphene oxide (GO), double-
oxidized graphene oxide (DGO), and GQDs to conjugate the hydrophobic
anticancer drug Cur separately. The interactions between Cur and the
­oxygen-containing functional groups of GOs, DGOs, and GQDs played a key
role in improving the Cur loading capacity. Since the number of oxygen-
containing functional groups on the surface of graphene derivatives was pH
dependent, the drug loading efficiency and release behavior were all pH
dependent. As-prepared GQD-Cur composites contained the highest
amount of Cur (40,800 mg/g) and exhibited the best anticancer activity
compared to the other composites including Cur alone at the same dose. In
this case, Cur and its GQDs composites had no fluorescence, and it was only
after the release of Cur from GQDs composites that the remaining GQDs
exhibited a fluorescence signal. Thus, the GQDs simultaneously acted as a
bioprobe for tumor imaging along with the release of the drug. Abdullah10
developed a way to achieve efficient and target specific delivery of GQDs car-
rying doxorubicin (DOX) using hyaluronic acid (HA) (GQD–HA) as a tar-
geting agent. Dopamine hydrochloride, which conjugated to HA, could be
easily bind to GQDs because of the catechol moieties. The particle size was
of ~20 nm, and non-toxic from MTT assay. They examined cytotoxic behav-
ior of DOX released from GQD–HA against CD44 overexpressed lung can-
cer A549 cells. The loading and release kinetics of the hydrophobic drug
DOX from GQDs under mildly acidic conditions showed that GQDs could

b2227_P2-V1_Ch-07.indd 117 23-Jun-17 2:57:21 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

118  Y. Chen & M. Chu

be considered as a novel drug carrier. Sui et al.22 developed a combination of

GQDs with cisplatin (CDDP) that can effectively enhance the cytotoxicity,
cell cycle arrest, and DNA fragmentation that was induced by CDDP, and
also their results showed an increase of chemotherapy efficacy of CDDP to
CDDP-resistant cells. The interaction of CDDP with DNA could be
improved by the GQDs and that was caused by the interaction of GQDs with
DNA.23–25 They believed that GQDs could improve the pharmaceutical per-
formance of CDDP via first increasing its cellular uptake, and then reinforc-
ing its interaction with DNA once inside cells.

3.1.2. GQDs as drug carriers and meanwhile for real-time monitoring

One of the advantages of GQDs as drug carrier is that they can realize real-
time monitoring of cellular uptake without the need for external dye. Wang26
and her colleagues prepared a folic acid (FA)-conjugated GQDs nanocom-
posites to load DOX and to realize sustained release. The loading efficiency
of DOX on GQD–FA was 68 ± 9 wt.%.The inherent stable fluorescence of
GQDs enabled real-time monitoring of the cellular uptake of the DOX–
GQD–FA nanocomposites and the consequent release of drugs (Figure 1).
These nanocomposites were specifically internalized rapidly by HeLa cells via
receptor-mediated endocytosis after only 30 min of incubation. DOX release
from GQD–FA and accumulation in cells could be detected after 8 h of incu-
bation. Since the GQDs could emit bright fluorescence, the time scale of the
fluorescence intensity changes in two channels were compared and the results
clearly showed that DOX–GQD–FA endocytosis was rapid while free DOX
accumulation was delayed and limited by the drug release step. Similarly, Qiu
et al.45 fabricated GQD-based traceable drug delivery system for the targeted,
pH-sensitive delivery of DOX into cancer cells. Arginine–glycine–aspartic
acid (RGD) peptides was used as the targeting moieties to enhance their
uptake efficacy by cancer cells. The amount of DOX contained within DOX–
GQDs and DOX–RGD–GQDs was 109 mg/mg and 101 mg/mg GQDs.
Since DOX–GQDs exhibited two fluorescence emission peaks around 480–
530 nm (excitation at 400 nm) and 550–630 nm (excitation at 499 nm),
both the drug carrier and the released drug could be tracked.
Wang28 developed a polyethylene glycol (PEG) grafted GQDs with a
strong green fluorescence for DOX delivery and real-time cell imaging. ­They
found that the photoluminescence (PL) of the as prepared GQDs–PEG were
size dependent, and the nanoparticles they obtained were with a mean diam-
eter of 15 nm. The PL quantum yield of the GQDs–PEG with 400 nm

b2227_P2-V1_Ch-07.indd 118 23-Jun-17 2:57:21 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Drug Delivery 119

Figure 1. Intracellular distribution of DOX–GQD–FA and free DOX. HeLa cells were incu-
bated with DOX–GQD–FA (33.7 mg/mL) at 37°C for (A) 0.5 h, (B) 8 h, and (C) 24 h. Left
to right columns: bright field s, GQD fluorescence, DOX fluorescence, overlay of the corre-
sponding. The GQDs and DOX were excited under 488 nm laser irradiation, and fluorescence
signals were collected from 500 to 530 nm and 552 to 617 nm, respectively. Reprinted with
permission from Ref. [26]. Copyright (2014) Elsevier.

excitation was about 18.8%, which was higher than other GQDs reported in
the literature. More importantly, the surface-passivated PEG on GQDs can
not only enhance PL intensity but also load drug by hydrogen bonding, and
improved the solubility of GQDs. Moreover, they investigated the loading
and release behavior of GQDs–PEG. High specific surface area endowed
them with high loading capability (2.5 mg/mg) to carry drug. The highest
loading capacity was observed at the neutral condition, rather than acidic or
­alkaline conditions (0.9 mg/mg at pH 5.5, 2.5 mg/mg at pH 7.4, and 1.1
mg/mg at pH 9). On the contrary, the release of DOX was different: 37% at
pH 7.4, 75%, and 51% at pH 5.5 and 9.0 after 48 h, more easily under acidic
and alkaline conditions.
In these three papers mentioned above, the loading and release behavior of
DOX were all pH dependent. And there are two main reasons for this, one is
attributed to the increased hydrophilicity and solubility of DOX at pH 5.0.29,30

b2227_P2-V1_Ch-07.indd 119 23-Jun-17 2:57:22 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

120  Y. Chen & M. Chu

The other is that the degree of hydrogen-bonding interaction between DOX

and the GQDs under acidic pH conditions is stronger than at neutral pH.31,32

3.1.3. GQDs as nuclear-targeted carriers

It is reported that there are plenty of nuclear pore complexes with a diam-
eter of 20–70 nm embedded in the nuclear membrane, which makes the
nuclear membrance impermeable to many probes, and particles smaller
than 10 nm can freely diffuse into the nucleus.33,34 Owing to the flexibility
and small size of the GQDs, it is very likely that drug-GQD conjugates can
penetrate the nucleus by diffusion and then improve the toxicity of drugs
to DNA. Some GQD-based nuclear-targeted drug carriers had been
reported. Wang15 revealed that small-sized GQDs could penetrate the
nucleus by diffusion and deliver DOX to the nucleus specifically, thereby
enhancing DNA cleavage activity of DOX markedly. These results showed
that the DOX-GQD conjugates migrated into nucleus more quickly than
DOX alone did. And under the same concentration of DOX, the DOX
accumulation in the nuclear of GQD–DOX and DOX alone was also dif-
ferent. GQDs could increase DOX nuclear uptake efficacy. They also found
that DNA cleavage enhancement by GQDs was time and concentration
dependent. The DNA cleavage increased if incubation time increased, and
was completed after 2 h. The DNA cleavage activity was different when the
concentration of GQDs changed, and reached the maximum with 150 μg/
mL of GQDs. This stable conjugates of GQD–DOX towards the nuclear
had the potential to improve the chemotherapy efficacy. Meanwhile,
Chen27 and his colleagues described a GQD-based Förster Resonant
Energy Transfer (FRET) system for nuclear-targeted DOX delivery. A pep-
tide TAT was used as the nuclear localization signal to transport the system
to the nucleus. The TAT was connected to GQDs through NH2–PFG–
NH2 as the bridge, and the whole system was denoted as DOX–GQDs–
PEG/TAT with an average diameter of 5 nm. In such a system, GQDs
simultaneously served as the carriers of drugs and donors of FRET pairs,
while drugs as the acceptors. The loading efficiency was 69.5% in a 2:1
ratio of DOX to GQDs–PEG/TAT. DOX–GQDs–PEG/TAT showed
higher toxicity to HeLa cells than DOX alone did. Habiba35 fabricated a
PEGylated silver nanoparticles decorated with GQDs as a platform to
deliver DOX into the nucleus by diffusion. They used this nanoparticle to
realize chemo-photodynamic therapy against HeLa and Prostate cancer
cell DU145 in vitro: the Ag-GQDs could efficiently deliver DOX and

b2227_P2-V1_Ch-07.indd 120 23-Jun-17 2:57:22 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Drug Delivery 121

meanwhile exhibited a cytotoxic effect due to the gen­eration of the reac-

tive singlet oxygen upon 425 nm irradiation. The singlet oxygen generated
by Ag-GQDs in aqueous media (i.e. PBS) was observed to be higher than
that of bare GQDs.
It has been demonstrated that DNA nucleobases and nucleotides inside
the nucleus can interact with the graphene sheet and a stronger interaction
than π–π stacking may be found between nucleobase oxygen with gra-
phene.36,37 So we may infer that GQDs also have this kind of property and
thus make themselves a perfect candidate for nucleus targeted drug
delivery.

3.2. GQDs-based carriers for gene delivery

GQDs could also be applied in gene delivery since their match size of biologi-
cal molecules and large surface area/loading capacity.38 There are three main
types of gene delivery systems39: viral vectors, non-viral vectors, and physical
delivery carriers. Viral vectors are efficient and most widely applied, however
they are impeded because of potential tumorigenicity and immunogenicity.40
Non-viral vectors like liposome and cationic polymers also have problems like
undesirable membrane destabilization and limited transfection and loading
efficiency.41 Physical delivery means using physical methods to deliver nucleic
acids into cells, for example, electroporation, genegun, and microneedles.40,42
This method is simple, however the nucleic acids delivered can be degraded
by nucleases easily.
GQDs belongs to the non-viral vectors but with high loading efficiency
and can be uptaken into the cytoplasm by endocytosis or membrane fusion
since their small size and biocompatibility.15,17 Dong et al.18 developed a mul-
tifunctional nanocomposite of poly (L-lactide) (PLA) and PEG-grafted
GQDs (f-GQDs) for simultaneous combined gene delivery to get enhanced
therapy and efficient intracellular microRNAs (miRNAs) imaging analysis.
The dysregulated expression of miRNAs is associated with various tumors and
Survivin can promote cellular mitosis and inhibit cellular apoptosis associated
with cancer by inhibiting apoptosis protein caspase-3 and caspase-7.43,44
Therefore, when the two gene probes targeting miRNA-21 and the Surviving
gene were loaded on the surface of the f-GQDs by specific adsorption and
π–π interaction between the probes and GQDs, they could induce inhibition
of cancer cell growth and more apoptosis (Figure 2). The uptake of a multi-
functional GQDs probe by HeLa cells can be monitored by the fluorescence
of GQDs.

b2227_P2-V1_Ch-07.indd 121 23-Jun-17 2:57:22 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

122  Y. Chen & M. Chu

Figure 2. Schematic illustration of f-GQDs for cell imaging and combined gene-targeting
agents delivery. Probe 1: the inhibitor probe of miRNA-21 and probe 2: Survivin antisense
oligodeoxynucleotide. Reprinted with permission from Ref. [18]. Copyright (2015) American
Chemical Society.

4. Discussion
Although until now not too many researches have been reported in the field
of GQDs based drug delivery systems, we can already see the special advan-
tages in them. Thanks to their excellent elec­trochemical and optical proper-
ties and large surface area, they can bind with a variety of aromatic
biomolecules, and may have the potential to monitor the whole process of
targeted drug delivery. Their small lateral size range enables GQDs to pene-
trate the nucleus by diffusion so as to enhance the toxicity of drugs to DNA
and gene delivery efficiency. Even though GQDs notably improve drug load-
ing efficacy by conjugation, they are not ideal for non-aromatic biomolecules,
and GQDs based composites are more promising in multifunctional drug
delivery. On the other hand, through the chemical binding, the drugs are
loaded on the surface, so they may not be as efficient in sustained and con-
trolled release as the 3D structure carriers. Also, there is still one problem we
need to pay attention to, which is the biosafety of GQDs. These carbon nano-
particles, like GQDs, may be non-toxicity to cells, but they are difficult to be
degraded and stay in the body for a long time. So, the long-term toxicity need
to be deeply studied for further usage.

References
1. Liang R, Wei M, Evans DG, Duan X. Inorganic nanomaterials for bioimaging, targeted
drug delivery and therapeutics. Chem Commun 2014; 50: 14071–14081.

b2227_P2-V1_Ch-07.indd 122 23-Jun-17 2:57:23 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Drug Delivery 123

2. Maurer N, Fenske DB, Cullis PR. Developments in liposomal drug delivery systems.
Expert Opin Biol Ther 2001; 1: 923–947.
3. Barenholz Y. Liposome application: problems and prospects. Curr Opin Colloid In 2001;
6: 66.
4. Miura Y, Takenaka T, Toh K, Wu S, Nishihara H, Kano MR, et al. Cyclic RGD-linked
polymeric micelles for targeted delivery of platinum anticancer drugs to glioblastoma
through the blood-brain tumor barrier. ACS Nano 2013; 7: 8583–8592.
5. Paciotti GFD, Kingston GI, Tamarkin L. Colloidal gold nanoparticles: A novel nanoparti-
cle platform for developing multifunctional tumor-targeted drug delivery vectors. Drug
Dev Res 2006; 67: 47–54.
6. Bacon M, Bradley SJ, Nann T. Graphene quantum dots. Part Syst Charact 2014; 31:
415–428.
7. Ding H, Wei J-S, Xiong H-M. Nitrogen and sulfur co-doped carbon dots with strong blue
luminescence. Nanoscale 2014; 6: 13817–13823.
8. Jiang F, Chen D, Li R, Wang Y, Zhang G, Li S, et al. Eco-friendly synthesis of size-­
controllable amine-functionalized graphene quantum dots with antimycoplasma properties.
Nanoscale 2013; 5: 1137.
9. Li Y, Zhao Y, Cheng H-H, Hu Y, Shi G-Q, Dai L-M, et al. Nitrogen-doped graphene
quantum dots with oxygen-rich functional groups. J Am Chem Soc 2012; 134: 15–18.
10. Abdullah-Al-Nahain, Lee J-E, In I, Lee H, Lee KD, Jeong JH, et al. Target delivery and
cell imaging using hyaluronic acid functionalized graphene quantum dots. Mol Pharm
2013; 10: 736–3744.
11. Huang C-L, Huang C-C, Mai F-D, Yen C-L, Tzing S-H, Hsieh HT, et al. Application
of paramagnetic graphene quantum dots as a platform for simultaneous dual-modality
bioimaging and tumor targeted drug delivery. J Mater Chem B 2015; 3: 651–664.
12. Yuan X, Liu Z, Guo Z, Ji Y, Jin M, Wang X. Cellular distribution and cytotoxicity of
graphene quantum dots with different functional groups. Nanoscale Res Lett 2014; 9:
1–9.
13. Zheng XT, Than A, Ananthanaraya A, Kim D-H, Chen P. Graphene quantum dots as
universal fluorophores and their use in revealing regulated trafficking of insulin receptors
in adipocytes. ACS Nano 2013; 7: 6278–6286.
14. Chandra A, Deshpande S, Shinde DB, Pillai VK, Singh N. Mitigating the cytotoxicity of
graphene quantum dots and enhancing their applications in bioimaging and drug delivery.
ACS Macro Lett 2014; 3: 1064–1068.
15. Wang C, Wu C, Zhou X, Han T, Xin X, Wu J, et al. Enhancing cell nucleus accumulation
and DNA cleavage activity of anti-cancer drug via graphene quantum dots. Sci Rep 2013;
3: 2852–2860.
16. Wang XY, Lei R, Huang HD, Wang N, Yuan L, Xiao RY, et al. The permeability and
transport mechanism of graphene quantum dots (GQDs) across the biological barrier.
Nanoscale 2015; 7: 2034–2041.
17. Shang W, Zhang X, Zhang M, Fan Z, Sun Y, Han M, et al. The uptake mechanism and
biocompatibility of graphene quantum dots with human neural stem cells. Nanoscale
2014; 6: 5799–5806.
18. Dong H, Dai W, Ju H, Lu H, Wang S, Xu L, et al. Multifunctional poly(L-lactide)-­
polyethylene glycol-grafted graphene quantum dots for intracellular MicroRNA imaging
and combined specific-gene-targeting agents delivery for improved therapeutics. ACS
Appl Mater Interfaces 2015; 7: 11015–11023.

b2227_P2-V1_Ch-07.indd 123 23-Jun-17 2:57:23 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

124  Y. Chen & M. Chu

19. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: Role of ATP-
dependent transporters. Nat Rev Cancer 2002; 2: 48–58.
20. Gottesman MM. Mechanism of cancer drug resistance. Annu Rev Med 2002; 53:
615–627.
21. Some S, Gwon AR, Hwang E, Bahn GH, Yoon Y, Kim Y, et al. Cancer therapy using
ultrahigh hydrophobic drug-loaded graphene derivatives. Sci Rep 2014; 3: 6314–6323.
22. Sui X, Luo C, Wang C, Zhang F, Zhang J, Guo S. Graphene quantum dots enhance
anticancer activity of cisplatin via increasing its cellular and nuclear uptake. Nanomedicine:
NBM 2016; 12: 1997–2006.
23. Zhou X, Zhang Y, Wang C, Wu X, Yang Y, Zheng B, et al. Photo-Fenton reaction of
graphene oxide: A new strategy to prepare graphene quantum dots for DNA cleavage.
ACS Nano 2012; 6: 6592–6599.
24. Zheng B, Wang C, Xin X, Liu F, Zhou X, Zhang J, et al. Electron transfer from graphene
quantum dots to the copper complex enhances its nuclease activity. J Phys Chem C 2014;
118: 7637–7642.
25. Zheng B, Wang C, Wu C, Zhou X, Lin M, Wu X, et al. Nuclease activity and cytotoxicity
enhancement of the DNA intercalators via graphene oxide. J Phys Chem C 2012; 116:
15839–15846.
26. Wang X, Sun X, Lao J, He H, Cheng T, Wang M, et al. Multifunctional graphene quan-
tum dots for simultaneous targeted cellular imaging and drug delivery. Colloid Surface B
2014; 122: 638–644.
27. Chen H, Wang Z, Zong S, Chen P, Zhu D, Wu L, et al. A graphene quantum dot-based
FRET system for nuclear-targeted and real-time monitoring of drug delivery. Nanoscale
2015; 7: 15477–15486.
28. Wang Z, Xia J, Zhou C, Via B, Xia Y, Zhang F, et al. Synthesis of strongly green photo-
luminescent graphene quantum dots for drug carrier. Colloid Surface B 2013; 112:
192–196.
29. Liu Z, Robinson JT, Sun XM, Dai HJ. PEGylated nanographene oxide for delivery of
water-insoluble cancer drugs. J Am Chem Soc 2008; 130: 10876–10877.
30. Liu Z, Sun XM, Nakayama-Ratchford N, Dai HJ. Supramolecular chemistry on water-
soluble carbon nanotubes for drug loading and delivery. ACS Nano 2007; 1: 50–56.
31. Bhirde AA, Patel V, Gavard J, et al. Targeted killing of cancer cells in vivo and in vitro
with EGF-directed carbon nanotube-based drug delivery. ACS Nano 2009; 3: 307–316.
32. Yang XQ, Grailer JJ, Rowland IJ, et al. Multifunctional stable and pH-responsive polymer
vesicles formed by hetero functional triblock copolymer for targeted anticancer drug
delivery and ultrasensitive MR imaging. ACS Nano 2010; 4: 6805–6817.
33. Pan L, He Q, Liu J, Chen Y, Ma M, Zhang L, et al. Nuclear-targeted drug delivery of
TAT peptide-conjugated monodisperse mesoporous silica nanoparticles. J AmChem Soc
2012; 134: 5722–5725.
34. Alber F, Dokudovskaya S, Veenhoff LM, Zhang W, Kipper J, Devos D, et al. The molecu-
lar architecture of the nuclear pore complex. Nature 2007; 450: 695–701.
35. Habiba K, Encarnacion-Rosado J, Garcia-Pabon K, Villalobos-Santos JC, Makarov VI,
Avalos JA, et al. Improving cytotoxicity against cancer cells by chemo-photodynamic
combined modalities using silver-graphene quantum dots nanocomposites. Int J Nanomed
2016; 11: 107–119.

b2227_P2-V1_Ch-07.indd 124 23-Jun-17 2:57:23 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Graphene Quantum Dots for Drug Delivery 125

36. Liu M, Zhao H, Chen S, Yu H, Quan X. Capture of double-stranded DNA in stacked-

graphene: Giving new insight into the graphene/DNA interaction. Chem Comm 2012;
48: 564–566.
37. Xu Z, Meher BR, Eustache D, Wang Y. Insight into the interaction between DNA bases
and defective graphenes: Covalent or non-covalent. J Mol Graph Model 2014; 47: 8–17.
38. Park K, Lee S, Kang E, Kim K, Choi K, Kwon IC. New generation of multifunctional
nanoparticles for cancer imaging and therapy. Adv Funct Mater 2009; 19: 1553−1566.
39. Chen W, Li H, Shi D, Liu ZG, Yuan W. Microneedles as a delivery system for gene
therapy. Front Pharmacol 2016; 7: 137.
40. McCaffrey J, McCrudden CM, Ali AA, Massey AS, McBride JW McCrudden MT, et al.
Transcending epithelial and intracellular biological barriers; A prototype DNA delivery
device. J Control Release 2016; 226: 238–47.
41. Noori-Zadeh A, Mesbah-Namin SA, Tiraihi T, Rajabibazl M, Taheri T. Nonviral human
proGDNF gene delivery to rat bone marrow stromal cells under ex vivo conditions.
J Neurol Sci 2014; 339: 81–86.
42. Somiari S, Glasspool-Malone J, Drabick JJ, Gilbert RA, Heller R, Jaroszeski MJ, et al.
Theory and in vivo application of electroporative gene delivery. Mol Ther 2000;
2: 178–87.
43. Altieri, DC. Validating Survivin as a cancer therapeutic. Nat Rev Cancer 2003; 3: 46−54.
44. Ryan BM, O’donovan N, Duffy MJ. Survivin: A new target for anti-cancer therapy.
Cancer Treat Rev 2009; 35: 553−562.
45. Qiu J, Zhang R, Li J, Sang Y, Tang W, Rivera Gil P, Liu H. Fluorescent graphene quan-
tum dots as traceable, pH-sensitive drug delivery systems. Int J Nanomed 2015; 10:
6709–6724.

b2227_P2-V1_Ch-07.indd 125 23-Jun-17 2:57:23 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Chapter 8

Toxicity of Graphene Quantum Dots

Maoquan Chu

Research Center for Translational Medicine at Shanghai East Hospital

150 Jimo Road, Shanghai 200120, P.R. China
School of Life Science and Technology, Tongji University
Shanghai 200092, P.R. China
[email protected]

Graphene quantum dots (GQDs) are novel biomaterials with numerous applications
in biomedical fields. Because these nanomaterials may be suitable for clinical use,
their bio-safety should be investigated carefully. Previous studies have focused on the
in vitro cytotoxicity of GQDs, showing that GQDs were biocompatible materials
and exhibited low cytotoxicity to living cells. However, animal studies may be more
useful for determining whether these nanomaterials can be used in humans. Several
research groups have investigated the in vivo toxicity of GQDs in mice, rats, and
zebrafish embryos. In this chapter, toxicity studies of GQDs are described along with
suggestions for future work.

1. Introduction
In the previous chapters, the physical and chemical properties of graphene
quantum dots (GQDs) have been described, as well as their biomedical appli-
cations. GQDs may be excellent carriers for drug delivery.1–5 These nanoma-
terials have also been used as biosensors in biomedical detection assays,6–16
and as photosensitizers and photothermal agents for cancer photodynamic
and photothermal therapies.17,18
GQDs have the largest specific surface area of all nanomaterials as they are
single-atom-thick materials and only 1–10 nm in lateral dimension size.
127

b2227_P2-V1_Ch-08.indd 127 23-Jun-17 2:57:45 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

128  M. Chu

Therefore, these ultrasmall nanosheets exhibit high-surface energy. Unmodified

GQDs or the GQDs without good modification tend to non-specifically bind to
surrounding materials, including cells and biomolecules, to release their surface
energy. The viability of cells adsorbed with a large quantity of nanomaterials may
decrease. In addition, ultrasmall nanoparticles may penetrate the blood–placenta
barrier and exert toxicity on the fetus. Therefore, the biosafety of GQDs should
be carefully investigated if these materials are to be used in clinical settings.
Until now, GQD toxicity studies have focused on cytotoxicity. Numerous
cells, including three different kinds of stem cells, neurospheres cells, pancreas
progenitor cells and cardiac progenitor cells,19 HeLa human cervical carci-
noma cells,3,20 A549 adenocarcinomic human alveolar basal epithelial
cells,3,21,22 oral epithelial carcinoma KB cells,21 breast cancer MDA–MB-231
cells,21 human neural glioma C6 cells,22 HEK293A normal human embryonic
kidney cells3 and rat pheochromocytoma PC12 cells,23 have been used to
measure the cytotoxicity of GQDs. All of these studies reported that GQDs
exhibited low toxicity to cells.
Studying the toxicity of GQDs to animals may be a more attractive pros-
pect for researchers because GQDs may hold clinical potential for in vivo
drug and gene delivery and cancer phototherapy. In recent years, in vivo
toxicities of graphene oxide (GO) and reduced GO (rGO) have been inten-
sively investigated.25–30 GQD is an important member of the graphene family.
However, the toxicity of GQD to animals has only been studied by a few
research groups,20,21,31,32 who used mouse,20,21 rat,21 and zebrafish31,32 as their
animal model. Mice and rats are mammals, and their genomes display high
homology with the human genome. Therefore, in this chapter, in vivo toxici-
ties of GQDs to mouse and rat are discussed.

2. In Vivo Biodistribution and Clearance of GQDs

Generally, we expect nanomaterials for in vivo applications to efficiently accu-
mulate in the target tissue and then, after completing their intended func-
tions, be rapidly removed from the body. GQDs are only several nanometers
in size and only one carbon atom in thickness; consequently, they may be
easily distributed throughout the body and then excreted.
Nurunnabi et al.21 investigated the toxicity of carboxylated GQDs to mice.
They prepared carboxylated GQDs using carbon fiber as a precursor. The
prepared GQDs were hexagonal with a lattice edge and 3–6 nm in size.
Because these GQDs emitted bright green fluorescence, they could be used
for ex vivo fluorescent imaging, and could also be used for superficial tumor
imaging. The carboxylated GQDs were injected into tumor-bearing mice via

b2227_P2-V1_Ch-08.indd 128 23-Jun-17 2:57:45 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Toxicity of Graphene Quantum Dots 129

tail veins for in vivo and ex vivo imaging studies. The tumors had been
grown from human breast cancer KB cells. GQD doses were 5 mg/kg and
10 mg/kg body weight. Tumor sites gradually emitted fluorescence 2 h post
injection; the brightest fluorescence was emitted 6–12 h post injection. Because
these GQDs have no red or near-infrared fluorescence (NIR), effects on major
organs such as lung and kidney could not be detected using this technique.
However, when the organs and tumors were isolated after sacrifice at 24 h post
injection, ex vivo imaging showed that the heart, liver, spleen, lung, kidney, and
tumor all emitted bright fluorescence in GQD-treated groups compared with
those in the untreated group. The tumor and major organ fluorescence in mice
treated with a 10 mg/kg dose was significantly brighter than that of mice
treated with a 5 mg/kg dose. These results indicate that GQDs could easily
distribute through blood and lymphatic capillaries and then arrive at various
organs. At 24 h post injection, the fluorescent signal at the tumor sites was
significantly decreased compared with fluorescence at 2–12 h post injection.
This finding implies that most GQDs may have been excreted from the body
by then, or that GQD fluorescence was not stable in mouse organs. They also
noted that fluorescence was brighter in tumors than in the major organs,
­indicating that the GQDs may have passively targeted the tumors through the
enhanced permeability and retention (EPR) effect of tumors.
To improve the sensitivity of in vivo fluorescent imaging, Chong et al.20
conjugated GQDs with a NIR fluorescent dye and then monitored their dis-
tribution in tumor-bearing mice. The GQDs had been synthesized by an
oxidative-cutting method using graphene as a precursor. These GQDs had a
sheet-like (planar) structure, and measured 3–5 nm in size and 0.5–1 nm in
thickness. These GQDs contained a large number of oxygen atoms (the
molar ratio of carbon to oxygen was 16:7). They then modified the GQDs
with polyethylene glycol (PEG) and conjugated them with Cy7 (a commonly
used NIR fluorescent dye) via amide bond for in vivo biodistribution analysis.
When the 4T1 breast cancer tumor-bearing mice were intravenously injected
with the GQD-PEG–Cy7 conjugate (15 mg/kg), fluorescent imaging
showed that the GQDs mainly accumulated in kidneys and tumors. At 48 h
post injection, both kidneys and tumors showed only weak fluorescence. This
finding implies that GQDs injected intravenously could passively target
tumor tissue, and are then excreted through the renal pathway. Similar results
were observed when tumor-bearing mice were intraperitoneally injected with
15 mg/kg of GQD-PEG–Cy7. The passive tumor targeting and fast clear-
ance of GQDs may have been a result of their small size.
GQDs may also be used as drug carriers delivered orally or transdermally.
However, in vivo biodistribution patterns of GQDs in mice after oral gavage

b2227_P2-V1_Ch-08.indd 129 23-Jun-17 2:57:45 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

130  M. Chu

or cutaneous penetration are not clear. Our research group30 recently demon-
strated that small (87.97 ± 30.83 nm) and large (472.08 ± 249.17 nm) rGO
nanomaterials accumulated mainly in the kidney of normal mice 1 and 15
days after gavage. Other organs, such as the stomach, lung, liver, and spleen,
also contained small amounts of rGO. Sixty days after the last gavage, most
of the rGO had been excreted renally. Although both rGO and GQDs are
graphene nanosheets, GQDs are significantly smaller than rGO. Therefore,
the in vivo biodistribution after oral gavage may be similar, but the excretion
rate of the GQDs should be faster. We found that 3-mercaptopropionic acid
capped-CdTe QDs several nanometers in diameter could penetrate mouse
skin and migrate into major organs.33 QD levels in spleen on days 1, 3, or 7
after skin staining were higher than those found in other organs, such as lung
and liver. QDs may have been excreted renally as the kidney contained
numerous cadmium atoms on days 3 and 7. GQDs are also several nanome-
ters in length, and their surfaces are also rich in carboxyl groups. Therefore,
GQDs may penetrate animal skin and distribute throughout the body, includ-
ing deposition in the spleen. These GQDs may be excreted in the urine.

3. Changes in Liver and Kidney Functions, and Hematological

Marker Levels
As GQDs can move through tissues and deposit in major organs at least 1 day
post injection, the health of GQD-treated animals should be seriously consid-
ered. Many diseases can be detected or predicted through blood analysis.
Because of hepatic first-pass effects and renal excretion of drugs, liver and
kidney toxicity of drugs should be investigated thoroughly before clinical
application.
Because the toxicity of a drug is related to the dose and duration of expo-
sure, Nurunnabi et al.21 intravenously injected carboxylated GQDs into rats
every 3 days for 22 days, for a total of seven injections. Dose groups received
5 or 10 mg/kg. Seven important hepatic indicators, namely albumin (ALB),
globulin (GLOB), glutamic pyruvate transaminase (GTP), alkaline phos-
phatase (ALP), glutamic oxaloacetic transaminase (GOT), total bilirubin
(TB), and total protein (TP) of the GQD-treated rats were measured periodi-
cally. All parameter levels in GQD groups were similar to those in the control
group, and all levels were within normal ranges. Two important indicators of
kidney function, blood urea nitrogen (BUN), and creatinine (CRE), were
also not significantly affected by GQD exposure. Five important hematology
markers — white blood cells (WBC), hemoglobin (HGB), hematocrit
(HCT), platelet count (PLT), and complete blood count (CBC) — were also

b2227_P2-V1_Ch-08.indd 130 23-Jun-17 2:57:45 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Toxicity of Graphene Quantum Dots 131

measured. None except WBC were significantly influenced by exposure to

GQDs 1 day post injection. In addition, all hematology markers in the GQD-
treated groups, including WBC, were similar to those in the control group on
days 8 and 22. It should be noted that WBC levels can be influenced by vari-
ous factors, including an inflammatory response, housing environment, food,
and water. WBC changes in GQD-treated rats may have been caused by one
of these factors. However, all hematology markers, including WBC, were still
within normal ranges. GQD-treated rats retained normal liver and kidney
functions and normal hematology marker levels.
Chong et al.20 treated mice with higher levels of GQDs for a long time.
Female Balb/c mice were intravenously or intraperitoneally injected with
20 mg/kg GQD–PEG every other day for 14 days, then monitored for up to
40 days. No mortality was observed, whereas a similar dose of GO–PEG
resulted in mortality of two-thirds of the mice. Six liver function markers —
ALB, ALP, GLOB, TP, aspartate transaminase (AST), and the albumin/
globulin ratio (A/G) — in GQD-treated mice were all within normal ranges,
as were two kidney function markers, BUN and CRE (Figure 1). Hematology
analysis also confirmed no adverse effects on mouse health from GQDs, as all
eight markers that were measured — WBC, PLT, HCT, HGB, red blood cell
(RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin
(MCH), and mean corpuscular hemoglobin concentration (MCHC) —
remained within normal ranges (Figure 2). These findings indicate that
GQDs modified with PEG and injected intravenously or intraperitoneally
may have no toxic effects on mouse liver, kidney, or hematological functions.
However, this is not to say that GQD exposure has no effect on mouse serum
biochemistry or blood marker levels, as there were some statistical differences

Figure 1. Changes in serum biochemistry levels of mice intravenously (i.v.) or intraperito-

neally (i.p.) injected with GQD–PEG, GO–PEG, or saline (control) 1 day post injection.
Reprinted with permission from Ref. 20. Copyright © 2014 Elsevier Ltd.

b2227_P2-V1_Ch-08.indd 131 23-Jun-17 2:57:46 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

132  M. Chu

Figure 2. Changes of hematology marker levels of mice intravenously (i.v.) or intraperito-

neally (i.p.) injected with GQD–PEG, GO–PEG, or saline (control) 1 day post injection.
Reprinted with permission from Ref. 20. Copyright © 2014 Elsevier Ltd.

in several parameters, such as ALT, BUN, and WBC, between the treated and
control groups.

4. Histological Structure Changes in Major Organs and Body

Weight Changes Following GQD Exposure
Compared with GO or rGO, GQDs are ultrasmall nanoparticles. Therefore,
injected GQDs can hardly be observed in mouse organs under an optical
microscope. In Nurunnabi et al.’s experiments, no dark dots could be found in
the mouse lung, liver, and other main organs using optical microscopy.21
However, in mice injected with GO or rGO, a large quantity of dark dots could
be observed clearly in the lung, liver, and spleen,20,26,27 representing aggrega-
tions of GO or rGO. Further, in Nurunnabi et al.’s experiments, no obvious
histopathological abnormalities or lesions could be observed in the mouse
heart, kidney, and spleen, but moderate pathological changes could be observed
in the liver and lung 21 days post injection of carboxylated GQDs, and con-
firmed by hematoxylin and eosin (H&E) staining.21 This finding deserves spe-
cial attention, as the liver and lung are important organs in mammals, and
higher concentrations of GQDs may be used in future clinical settings.
However, Chong et al.20 reported that histological structures of mouse
organs, including liver and lung, were not influenced following seven injec-
tions (20 mg/kg of GQDs–PEG for each mouse every other day). H&E
staining confirmed that organs in the GQDs–PEG group were similar to
those in the control group.
Body weight can serve as a measure of the low or nearly no toxicity of
GQD. The studies cited earlier found that fluctuations in mouse body weight

b2227_P2-V1_Ch-08.indd 132 23-Jun-17 2:57:46 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Toxicity of Graphene Quantum Dots 133

in the GQD-exposed groups were similar to those in control groups.20,21 For

example, when mice were injected with GQD–PEG (20 mg/kg) every alter-
nate day for 14 days, body weights increased from an initial 21 to 29 g over
a period of about 55 days.20 There was no statistical difference in body weight
changes between the GQD groups and the control group.

5. Phototoxicity of GQDs to Bacteria and Mouse-Borne Tumors

As mentioned above, intravenously or intraperitoneally injected GQDs dis-
played low or nearly no toxicity to mice or rats. However, when GQDs are
irradiated by a laser, they may exert toxicity on cells and tissues. GQDs have
a broad absorption spectrum, ranging from ultraviolet (UV) to red or NIR
wavelengths. Irradiation with a laser at a suitable wavelength would cause
GQDs to generate reactive oxygen species (ROS).17,18,34 ROS such as •OH
and 1O2 are toxic to biomolecules, such as DNA, and would induce cell apop-
tosis or necrosis.
In 2014, Ristic BZ et al. reported that the GQDs could destroy bacterial
cells under blue light irradiation.35 The GQDs used by Ristic BZ et al. were
prepared by a electrochemical method and were 20–67 nm in diameter and
~ 3 nm in height. They then used a light in a wavelength ranging from 465 to
475 nm (power: 1 W) to trigger the GQDs to kill two strains of pathogenic
bacteria, methicillin-resistant Staphylococcus aureus and Escherichia coli. The
results showed that the viabilities of bacterial cells after being simultaneously
treated with the GQDs and blue light significantly decreased compared with
those of bacterial cells treated with GQDs or blue light alone. The cytotoxic
effect of GQD on bacterial was dependent on GQD concentration and the
duration of photoexposure. The reduced viabilities of the bacterial were
mainly caused by the ROS preduced by the blue light-triggered GQDs. These
indicated that GQDs could be used as antibacterial agents.
Markovic et al.34 found that electrochemically produced GQDs, upon
irradiation with a 470-nm laser (power, 1W), could efficiently produce singlet
oxygen (1O2) in a cell culture. U251 human glioma cells incubated with
GQDs (200 mg/mL) absent laser irradiation exhibited high viability.
However, the photodynamic cytotoxicity of the GQDs after 10 min of irra-
diation killed half of the cells. Ge et al.18 prepared GQDs using polythiophene
derivatives as the carbon source, and demonstrated that these GQDs generate
1
O2 upon irradiation with a white light (400–800 nm) (power density: 6.5
mW/cm2). The 1O2 quantum yield of this GQD solution under laser irradia-
tion at different excitation wavelengths (538, 549, and 562 nm) was almost
consistent (approximately 1.3), and 1O2 produced by the irradiated GQDs
was toxic to HeLa cells.

b2227_P2-V1_Ch-08.indd 133 23-Jun-17 2:57:46 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

134  M. Chu

Tumors may be significantly damaged by the phototoxicity of GQDs. Ge

et al.18 injected GQDs (4 mg/kg) into tumor-bearing mice to investigate
whether tumor growth was inhibited by the phototoxicity of GQDs. The
tumors containing GQDs were irradiated for 10 min with a white light
(400–800 nm) at a power density of 80 mW/cm2 twice on the first and sev-
enth days. The tumors began to decompose after 9 days, and were destroyed
after 17 days. No tumors re-grew over the course of 50 days. In controls,
tumor growth was not noticeably affected by treatment with GQDs alone or
light irradiation alone.
Upon laser irradiation, GQDs can also convert laser light energy into
heat.17,18 Our research group17 demonstrated that the temperature of a GQD
aqueous solution (2.5 mg/mL) increased from 25.1 ± 0.1 to 42.6 ± 0.2°C
after exposure of 20 min to 671 nm laser irradiation (0.2 W/cm2). This pho-
tothermal conversion effect may not only destroy cancer cells, but may also
increase GQD photodynamic toxicity.
The phototoxic property of GQDs is used in local cancer photodynamic
and photothermal therapies. Only tumors or other tissues requiring treatment
would be damaged by light irradiation when containing high concentrations
of GQDs. GQD phototoxicity may not induce systemic toxicity.

6. Discussion
Recent studies have reported that GQDs exert little or no effect on mouse or
rat health. One reason may be their small size and unique carbon-based com-
position. Ultrasmall GQDs are easily excreted from the murine body. This
may be a common character of ultrasmall nanoparticles. Choi et al.36 reported
that semiconductor QDs with no more than 5.5 nm in final hydrodynamic
diameter were excreted efficiently in the urine of rats and mice.
As mentioned earlier, the toxicity of nanomaterials is related to their dose.
Wang et al.31 incubated zebrafish embryos with GQDs (0, 12.5, 25, 50, 100,
and 200 mg/mL) for 4–96 h post fertilization. Low concentrations of GQDs
were non-toxic to the zebrafish embryos. However, the hatching and heart
rates decreased, and mortality in the embryos increased in a concentration-
dependent manner. Various embryonic malformations, including pericardial
edema, vitelline cyst, bent spine, and bent tail, were observed when GQD
concentrations were increased to 200 mg/mL. In published studies, the high-
est dose of GQDs used in mice was 20 mg/kg every other day for 14 days.
The toxicity of GQDs in higher concentrations (>20 mg/kg) to mammals is
unknown, and should be investigated.
The toxicity of nanomaterials to animals is also related to their surface
potentials and the properties of ligand molecules used to modify

b2227_P2-V1_Ch-08.indd 134 23-Jun-17 2:57:46 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Toxicity of Graphene Quantum Dots 135

the nanomaterials.22,37 Yuan et al.22 investigated the cytotoxicity of GQDs

modified with three functional groups [NH2, COOH, and CO–N (CH3)2] in
human A549 lung carcinoma cells and neural glioma C6 cells. GQDs with all
three surface chemical groups exhibited low toxicity to cells. In addition, the
cytotoxicity of GQDs with CO–N (CH3)2 (100 or 200 mg/mL) was lower
than that of GQDs with NH2 and COOH in in both cell types. In A549 cells,
cytotoxicity of GQD–NH2 was lower than that of GQD–COOH. The toxic-
ity of the GQDs with different surface ligands to animals is unknown.
Because GQDs are only several nanometers in size, whether these nano-
particles can penetrate across the blood–placenta barrier and blood–testis bar-
rier is unclear. No reports on the reproductive toxicity of GQDs in mammals
have been published to date. GQDs possess great potential for clinical drug
delivery and cancer therapy applications, and the corresponding in vivo toxic-
ity should be investigated further.

References
1. Lv O, Tao Y, Qin Y, Chen C, Pan Y, Deng L, Liu L, Kong Y. Highly fluorescent and
morphology-controllable graphene quantum dots-chitosan hybrid xerogels for in vivo imag-
ing and pH-sensitive drug carrier. Mater Sci Eng C Mater Biol Appl 2016; 67: 478–486.
2. Justin R, Tao K, Román S, Chen D, Xu Y, Geng X, Ross IM, Grant RT, Pearson A,
Zhou G, MacNeil S, Sun K, Chen B. Photoluminescent and superparamagnetic
reduced graphene oxide–iron oxide quantum dots for dual-modality imaging, drug
­delivery and photothermal therapy. Carbon 2016; 97: 54–70.
3. Wang X, Sun X, Lao J, He H, Cheng T, Wang M, Wang S, Huang F. Multifunctional
graphene quantum dots for simultaneous targeted cellular imaging and drug delivery.
Colloid Surface B 2014; 122: 638–644.
4. Wang Z, Xia J, Zhou C, Via B, Xia Y, Zhang F, Li Y, Xia L, Tang J. Synthesis of strongly
green-photoluminescent graphene quantum dots for drug carrier. Colloid Surface B
2013; 112: 192–196.
5. Sui X, Luo C, Wang C, Zhang F, Zhang J, Guo S. Graphene quantum dots enhance
anticancer activity of cisplatin via increasing its cellular and nuclear uptake. Nanomed-
Nanotechnol 2016; 7: 1997–2006.
6. Wang L, Tricard S, Yue P, Zhao J, Fang J, Shen W. Polypyrrole and graphene quantum
dots @ Prussian Blue hybrid film on graphite felt electrodes: Application for amperometric
determination of L-cysteine. Biosens Bioelectron 2016; 77: 1112–1118.
7. Zhou X, Jiang Y, Li Z, Gu Z, Wang G. Improved activity and thermo-stability of the horse
radish peroxidase with graphene quantum dots and its application in fluorometric detec-
tion of hydrogen peroxide. Spectrochim Acta A 2016; 165: 106–113.
8. Hu T, Zhang L, Wen W, Zhang X, Wang S. Enzyme catalytic amplification of miRNA-155
detection with graphene quantum dots-based electrochemical biosensor. Biosens
Bioelectron 2016; 77: 451–456.
9. Vasilescu I, Eremia SAV, Kusko M, Radoi A, Vasile E, Radu G-L. Molybdenum disulphide
and graphene quantum dots as electrode modifiers for laccase biosensor. Biosens
Bioelectron 2016; 75: 232–237.

b2227_P2-V1_Ch-08.indd 135 23-Jun-17 2:57:46 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

136  M. Chu

10. Poon C-Y, Li Q, Zhang J, Li Z, Dong C, Lee AW-M, Chan W-H, Li H-W. FRET-based
modified graphene quantum dots for direct trypsin quantification in urine. Anal Chim
Acta 2016; 917: 64–70.
11. Liang R-P, Qiu W-B, Zhao H-F, Xiang C-Y, Qiu J-D. Electrochemiluminescence reso-
nance energy transfer between graphene quantum dots and graphene oxide for sensitive
protein kinase activity and inhibitor sensing. Anal Chim Acta 2016; 904: 58–64.
12. Nirala NR, Abraham S, Kumar V, Bansal A, Srivastava A, Saxena PS. Colorimetric detec-
tion of cholesterol based on highly efficient peroxidase mimetic activity of graphene
quantum dots. Sensor Actuatr B-Chem 2015; 218: 42–50.
13. Shi J, Chan C, Pang Y, Ye W, Tian F, Lyu J, Zhang Y, Yang M. A fluorescence resonance
energy transfer (FRET) biosensor based on graphene quantum dots (GQDs) and gold
nanoparticles (AuNPs) for the detection of mecA gene sequence of Staphylococcus aureus.
Biosens Bioelectron 2015; 67: 595–600.
14. He Y, Wang X, Sun J, Jiao S, Chen H, Gao F, Wang L. Fluorescent blood glucose monitor
by hemin-functionalized graphene quantum dots based sensing system. Anal Chim
Acta 2014; 810: 71–78.
15. Razmi H, Mohammad-Rezaei R. Graphene quantum dots as a new substrate for immo-
bilization and direct electrochemistry of glucose oxidase: Application to sensitive glucose
determination. Biosens Bioelectron 2013; 41: 498–504.
16. Lu J, Yan M, Ge L, Ge S, Wang S, Yan J, Yu J. Electrochemiluminescence of blue-lumi-
nescent graphene quantum dots and its application in ultrasensitive aptasensor for adeno-
sine triphosphate detection. Biosens Bioelectron 2013; 47: 271–277.
17. Wo F, Xu R, Shao Y, Zhang Z, Chu M, Shi D, Liu S. Multimodal cancer theraputics via
magneto-mechanical force and near-infrared laser-triggered doxorubicin-loaded graphene
quantum dot-coated hollow magnetic nanospheres. Theranostics 2016; 6: 485–500.
18. Ge J, Lan M, Zhou B, Liu W, Guo L, Wang H, Jia Q, Niu G, Huang X, Zhou H,
Meng X, Wang P, Lee C-S, Zhang W, Han X. A graphene quantum dot photodynamic
therapy agent with high singlet oxygen generation. Nat Commun 2014; 5: 4596.
19. Zhang M, Bai L, Shang W, Xie W, Ma H, Fu Y, Fang D, Sun H, Fan L, Han M, Liu C,
Yang S. Facile synthesis of water-soluble, highly fluorescent graphene quantum dots as a
robust biological label for stem cells. J Mater Chem 2012; 22: 7461–7467.
20. Chong Y, Ma Y, Shen H, Tu X, Zhou X, Xu J, Dai J, Fan S, Zhang Z. The in vitro and
in vivo toxicity of graphene quantum dots. Biomaterials 2014; 35: 5041–5048.
21. Nurunnabi M, Khatun Z, Huh KM, Park SY, Lee DY, Cho KJ, Lee Y-k. In Vivo biodis-
tribution and toxicology of carboxylated graphene quantum dots. ACS Nano 2013; 7:
6858–6867.
22. Yuan X, Liu Z, Guo Z, Ji Y, Jin M, Wang X. Cellular distribution and cytotoxicity of
graphene quantum dots with different functional groups. Nanoscale Res Lett 2014;
9: 108.
23. Liu Y, Xu L-P, Dai W, Dong H, Wen Y, Zhang X. Graphene quantum dots for the inhibi-
tion of β amyloid aggregation. Nanoscale 2015; 7: 19060–19065.
24. Zhang XY, Yin JL, Peng C, Hu WQ, Zhu ZY, Li WX, et al. Distribution and biocompat-
ibility studies of graphene oxide in mice after intravenous administration. Carbon 2011;
49: 986–95.
25. Fu CH, Liu TL, Li LL, Liu HY, Liang QH, Meng XW. Effects of graphene oxide on the
development of offspring mice in lactation period. Biomaterials 2015; 40: 23–31.

b2227_P2-V1_Ch-08.indd 136 23-Jun-17 2:57:46 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Toxicity of Graphene Quantum Dots 137

26. Yang K, Gong H, Shi XZ, Wan JM, Zhang YJ, Liu Z. In vivo biodistribution and toxicol-
ogy of functionalized nano-graphene oxide in mice after oral and intraperitoneal adminis-
tration. Biomaterials 2013; 34: 2787–2795.
27. Yang K, Wan JM, Zhang SA, Zhang YJ, Lee ST, Liu Z. In vivo pharmacokinetics, long-
term biodistribution, and toxicology of PEGylated graphene in mice. ACS Nano 2011; 5:
516–22.
28. Xu S, Zhang Z, Chu M. Long-term toxicity of reduced graphene oxide nanosheets:
Effects on female mouse reproductive ability and offspring development. Biomaterials
2015; 54: 188–200.
29. Liang S, Xu S, Zhang D, He J, Chu M. Reproductive toxicity of nanoscale graphene oxide
in male mice. Nanotoxicology 2015; 19: 92–105.
30. Zhang D, Zhang Z, Liu Y, Chu M, Yang C, Li W, Shao Y, Yue Y, Xu R. The short- and
long-term effects of orally administered high-dose reduced graphene oxide nanosheets on
mouse behaviors. Biomaterials 2015; 68: 100–113.
31. Wang ZG, Zhou R, Jiang D, Song JE, Xu Q, Si J, Ping CY, Xin Z, Lu G, Zhen LJ, Hong
Z, Bin L. Toxicity of Graphene Quantum Dots in Zebrafish Embryo. Biomed Environ Sci
2015; 28: 341–351.
32. Huang CL, Huang CC, Mai FD, Yen CL, Tzing SH, Hsieh HT, Ling YC, Chang JY.
Application of paramagnetic graphene quantum dots as a platform for simultaneous dual-
modality bioimaging and tumor-targeted drug delivery. J Mater Chem B 2015; 3:
651–664.
33. Chu M, Wu Q, Wang J, Hou S, Miao Y, Peng J, Sun Y. In vitro and in vivo transdermal
delivery capacity of quantum dots through mouse skin. Nanotechnology 2007; 18: 455103
1–6.
34 Markovic ZM, Ristic BZ, Arsikin KM, Klisic DG, Harhaji-Trajkovic LM, Todorovic-
Markovic BM, Kepic DP, Kravic-Stevovic TK, Jovanovic SP, Milenkovic MM,
Milivojevic DD, Bumbasirevic VZ, Dramicanin MD, Trajkovic VS. Graphene quantum
dots as autophagy-inducing photodynamic agents. Biomaterials 2012; 33: 7084–7092.
35. Ristic BZ, Milenkovic MM, Dakic IR, Todorovic-Markovic BM, Milosavljevic MS,
Budimir MD, Paunovic VG, Dramicanin MD, Markovic ZM, Trajkovic VS. Photodynamic
antibacterial effect of graphene quantum dots. Biomaterials 2014; 35: 4428–4435.
36. Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, Bawendi MG, Frangioni JV.
Renal clearance of quantum dots. Nat Biotechnol 2007; 25: 1165–1170.
37. Bozich JS, Lohse SE, Torelli MD, Murphy CJ, Hamers RJ, Klaper RD. Surface chemistry,
charge and ligand type impact the toxicity of gold nanoparticles to Daphnia magna.
Environ Sci Nano 2014; 1: 260–270.

b2227_P2-V1_Ch-08.indd 137 23-Jun-17 2:57:46 PM

 b2227-P2-V1   Synthesis and Biomedical Applications of Graphene Quantum Dots

Index

A I
amino-functionalization, 19–20, 29–31 in vivo biodistribution, 128–130
in vivo clearance, 128
B in vivo imaging, 99, 107, 109–110
bottom-up strategies, 7, 11
M
C medical and pharmaceutical analyses,
cancer cellular imaging, 99–101 57–58
chemotherapy drug delivery, 117 microwave-solvothermal method, 19,
chromatographic detection, 89 21, 28, 37
controllable synthesis, 13
O
D optical detection, 83
doping, 39, 44–45, 47, 52
P
E photoluminescence, 19
electrochemical detection, 89 phototoxicity, 133–134
plasmons, 39–44, 48–49
F
fluorescence imaging, 97–98, 107, S
109–110 sensor, 57–58, 60–61, 66, 71
food analysis, 77–80, 83–84 stem cellular imaging, 102–103, 110

G T
gene delivery, 121–122 top-down strategies, 7–8
graphene quantum dots, 1, 19–20, 39, toxicity, 127–128, 130, 132–135
57, 64, 77, 80, 97, 115–116, 127 tunable fluorescent properties, 4

139

b2227_P2-V1_Index.indd 139 23-Jun-17 2:58:33 PM