nanomaterials

Article
Synthesis of Carbon Quantum Dot Nanoparticles
Derived from Byproducts in Bio-Refinery Process
for Cell Imaging and In Vivo Bioimaging
Caoxing Huang 1,† , Huiling Dong 2,† , Yan Su 1 , Yan Wu 2 , Robert Narron 3 and Qiang Yong 1, *
1 Jiangsu Co-Innovation Center for Efficient Processing and Utilization of Forest Resources,
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China;
hcx@njfu.edu.cn (C.H.); suyan1994@njfu.edu.cn (Y.S.)
2 College of Furnishings and Industrial Design, Nanjing Forestry University, Longpan Road 159,
Nanjing 210037, China; ll233d@163.com (H.D.); wuyan@njfu.edu.cn (Y.W.)
3 Department of Forest Biomaterials, North Carolina State University, Campus Box 8005, Raleigh,
NC 27695-8005, USA; robnarron@gmail.com
* Correspondence: swhx@njfu.com.cn; Tel.: +86-025-8542-7797
† Huiling Dong contributed equally to this work, regarding as the first author.




Received: 17 February 2019; Accepted: 4 March 2019; Published: 7 March 2019


Abstract: The carbon quantum dot (CQD), a fluorescent carbon nanoparticle, has attracted
considerable interest due to its photoluminescent property and promising applications in cell
imaging and bioimaging. In this work, biocompatible, photostable, and sustainably sourced CQDs
were synthesized from byproducts derived from a biorefinery process using one-pot hydrothermal
treatment. The main components of byproducts were the degradation products (autohydrolyzate)
of biomass pretreated by autohydrolysis. The as-synthesized CQDs had a size distribution from
2.0–6.0 nm and had high percentage of sp2 and sp3 carbon groups. The CQDs showed blue-green
fluorescence with a quantum yield of ~13%, and the fluorescence behaviors were found to be stable
with strong resistance to photobleaching and temperature change. In addition, it is found that
the as-synthesized CQDs could be used for imaging of cells and tumors, which show potential
applications in bioimaging and related fields such as phototherapy and imaging.

Keywords: carbon quantum dots; autohydrolyzate; hydrothermal treatment; fluorescence;

bioimaging

1. Introduction
The carbon quantum dot (CQD), a quantum-sized carbon material, has drawn great
attention as a novel material due to its super hydrophilicity, good biocompatibility, and excellent
photoluminescence properties [1–4]. The light-absorbing properity of CQD render them applicable
for photocatalysis, energy conversion, optoelectronic devices, and chemical sensors [5–7]. In addition,
the low toxicity associated with CQD enables them to be harmlessly implemented in instances of cell
imaging, in vivo biomaging, and drug delivery systems [4].
Generally, CQD can be manufactured by a “top-down” route from different carbon-based
precursors (such as graphite, carbon fibers, and biological sources) [8–10]. Strategies for preparing
CQD from such materials always involve both chemical oxidation and solvothermal treatment under
harsh conditions. Such conditions are necessary in order to oxidize, exfoliate, and diminish the carbon
precursors into nanoscale carbonaceous matter. Concentrated acid (such as sulfuric acid and nitric
acid) and time-intensive treatment are required to establish the harsh conditions necessary for CQD
formulation [11–13]. However, these methods are not regarded as green because of the personal

Nanomaterials 2019, 9, 387; doi:10.3390/nano9030387 www.mdpi.com/journal/nanomaterials

personal safety and environmental hazards associated with the chemistry applied. In an attempt to
establish a green and facile process, hydrothermal “bottom-up” methods have attracted growing
attention due to this method’s lessened processing severity. The term “bottom-up” refers to
synthesis of CQD
Nanomaterials from
2019, 9, 387 molecular precursors such as citric acid, glucose, xylan, or resin2 of [14–16].
11

Hydrothermal synthesis of CQD is a low-cost, facile, rapid, and green method [13]. However, there
remains
safetyaand challenge
environmental regarding
hazards identifying
associated withand thesourcing
chemistry suitable
applied. and
In an inexpensive bio-based
attempt to establish
precursors.
a green and facile process, hydrothermal “bottom-up” methods have attracted growing attention due
From
to the precursors
this method’s investigated
lessened processing severity.across
The term recent literature,
“bottom-up” refers toit synthesis
can beof CQD inferredfrom that
monosaccharides (glucose, xylose, others) from biomass is a renewable and possible precursor
molecular precursors such as citric acid, glucose, xylan, or resin [14–16]. Hydrothermal synthesis of to
produce the CQD. Lignocellulosic biorefineries can produce monosaccharides by different
CQD is a low-cost, facile, rapid, and green method [13]. However, there remains a challenge regarding
identifying[17,18].
technologies and sourcing
During suitable and inexpensive
a biorefinery process,bio-based precursors.
pretreatment is an essential step required to
From the precursors investigated across recent literature, it can be inferred that monosaccharides
disrupt the indurative structure of biomass for subsequent enzymatic hydrolysis [19,20]. One
(glucose, xylose, others) from biomass is a renewable and possible precursor to produce the
pretreatment technology, autohydrolysis, is remarkably green and effective from both a
CQD. Lignocellulosic biorefineries can produce monosaccharides by different technologies [17,18].
technological and an economic
During a biorefinery process,perspective.
pretreatmentAutohydrolyis
is an essential stepis able to release
required ~10–20%
to disrupt of glucan and
the indurative
~50%structure
of xylan from for
of biomass biomass
subsequent into enzymatic
an aqueous carbohydrate-rich
hydrolysis hydrolysate,
[19,20]. One pretreatment termed as
technology,
autohydrolyzate
autohydrolysis, [19]. Because thegreen
is remarkably monosaccharide
and effective concentrations in the autohydrolyzate
from both a technological and an economic are low
and perspective.
the solution also contains
Autohydrolyis is able toa release
wide ~10–20%
variety ofofglucan lignin-derived
and ~50% of phenolic
xylan from compounds,
biomass
autohydrolyzate
into an aqueous is often disregarded as
carbohydrate-rich a biorefinery
hydrolysate, waste
termed as stream [21]. Hence,
autohydrolyzate we Because
[19]. tried to the pursue
production of CQD from the monosaccharides present in autohydrolyzate. A significant field of
monosaccharide concentrations in the autohydrolyzate are low and the solution also contains a wide
research
varietyaround CQD is their
of lignin-derived phenolicfunctionalization for bioimaging.
compounds, autohydrolyzate is oftenOne report demonstrated
disregarded as a biorefinery that
waste stream [21]. Hence, we tried to pursue production of
amino-passivated CQD exhibited strong optical absorption for cell imaging and in vivo biomagingCQD from the monosaccharides
present in autohydrolyzate. A significant field of research around CQD is their functionalization for
[22]. The application for biomaging was attributed to the enrichment of amine groups at the surfaces
bioimaging. One report demonstrated that amino-passivated CQD exhibited strong optical absorption
of the CQD. Amine-based functionalization agents, such as 1,2-ethylenediamine, trimethylamine,
for cell imaging and in vivo biomaging [22]. The application for biomaging was attributed to the
poly(ethylenemine),
enrichment of amine and butanediamine,
groups at the surfaces of have beenAmine-based
the CQD. applied for CQD functionalization
functionalization agents, such as across
various works [23–25]. Urea
1,2-ethylenediamine, is a low-cost,
trimethylamine, low toxicity, andand
poly(ethylenemine), lowbutanediamine,
corrosivity doped have agent, which has
been applied
beenforused
CQD forfunctionalization
surface functionalization
across various of bottom-up
works [23–25].CQD prepared
Urea from sodium
is a low-cost, citrate
low toxicity, andandlowcitric
acid corrosivity
[26]. However, no work has shown if the various components in the
doped agent, which has been used for surface functionalization of bottom-up CQD prepared autohydrolyzate can be
dopedfrombysodium
urea citrate
to prepare
and citric amino-passivated
acid [26]. However,CQD no work forhasbiomaging,
shown if theusing variousthe green one-pot
components in
hydrothermal treatment as synthesis method. Meanwhile, utilizing the biorefinery byproducts as
the autohydrolyzate can be doped by urea to prepare amino-passivated CQD for biomaging, using the
the precursor
green one-pot to synthesize
hydrothermal CQDtreatment
can potentially showmethod.
as synthesis the synthesis process
Meanwhile, is lowthe
utilizing cost.
biorefinery
byproducts as the precursor to synthesize CQD can potentially show
In this work, in order to evaluate whether the byproducts in bio-refinery process can the synthesis process is lowbecost.
used as
In this work, in order to evaluate whether the byproducts in bio-refinery process can be used
the precursors to synthesize CQD, different renewable biomass of wheat straw (WS) and bamboo
as the precursors to synthesize CQD, different renewable biomass of wheat straw (WS) and bamboo
residues (BR) were used as the raw materials to prepare the autohydrolyzate. The main reactants in
residues (BR) were used as the raw materials to prepare the autohydrolyzate. The main reactants
the autohydrolyzate are glucose and xylose, which were further mixed with urea to prepare
in the autohydrolyzate are glucose and xylose, which were further mixed with urea to prepare
amino-passivated
amino-passivated CQDs CQDs (CQD-WS
(CQD-WS(wheat (wheatstraw) and CQD-BR
straw) and CQD-BR(bamboo (bamboo residues))
residues)) using
using one-pot
one-pot
hydrothermal
hydrothermal treatment.
treatment.The The optic
optic andandphysicochemical
physicochemical properties
properties of the of the as-synthesized
as-synthesized CQDs were CQDs
wereinvestigated
investigated andand theirtheir potential
potential applications
applications for cell and
for cell imaging imaging
in vivoand in vivo were
bioimaging bioimaging
explored,were
explored,
as shownas shown
in Scheme in 1.
Scheme 1. The
The novelty of novelty
this workof this the
is that work is that the
synthesized CQDs synthesized
from byproductsCQDsinfrom
byproducts
bio-refineryin process
bio-refinery process
can indeed canasindeed
function function
agents for as agents
cell imaging for cell
and in vivo imaging
bioimaging, andhave
which in vivo
bioimaging, which have never been reported before.
never been reported before.

CH2OH
H OH
O
H
OH H
Hydrothermal UV On
OH H
Reaction
H OH
H In vivo bioimaging for tumor
OH
180 oC, 4 h
H O
H H
OH

OH H
H OH
Cell Imaging
Byproducts in bio-
refinery process

H2N C NH2

Scheme 1. The illustration of the green route for carbon quantum dots (CQDs) synthesis from
Scheme 1. The illustration of the green route for carbon quantum dots (CQDs) synthesis from
biorefinery byproducts as the precursor.
biorefinery byproducts as the precursor.
Nanomaterials 2019, 9, 387 3 of 11

2. Materials and Methods

2.1. Materials
Wheat straw and bamboo residues were used as raw materials to prepare autohydrolyzate.
The bamboo residues used were provided by the He Qi Cang Bamboo Processing Factory (Fujian,
China). Wheat straw was obtained from farm land in Jiangsu province, China.

2.2. Synthesis of Carbon Quantum Dot (CQD) Using One-Pot Hydrothermal Treatment
Autohydrolyzates (AH) from wheat straw and bamboo residues were prepared according to our
previous work [19], termed AH-WS and AH-BR. Vacuum concentration was applied to the original
autohydrolyzate to increase the final concentration of monosaccharides (glucose, xylose, and arabinose)
in the AH-WS and AH-BR to ~40 g/L.
One-pot hydrothermal treatment was carried out in a 100 mL bomb heated by a hot oil bath.
Inside the bomb, 50 mL of concentrated AH-WS and AH-BR (with 2 g total monosaccharides) was
mixed with 6 g urea and heated at 180 ◦ C for 4 h. After time, the bomb was carefully removed from the
oil bath and immediately cooled in ice water. After opening the bombs, the liquid and precipitate were
separated by centrifugation at 10,000× g for 10 min. The liquid was then dialyzed using a dialysis
tube (molecular weight cutoff of 8000 g/mol). The material remaining outside of the dialysis tube was
denoted as the carbon quantum dots (CQD). The CQD obtained from the autohydrolyzates of AH-WS
and AH-BR were termed as CQD-WS and CQD-BR. The CQD-WS and CQD-BR solutions were finally
dried by vacuum to produce CQD powders.

2.3. Characterization of the Synthesized CQDs

The Fourier transform–infrared (FT–IR) spectra of the CQD samples were recorded on a FT–IR
spectrophotometer (VERTEX 80V, Karlsruhe, Germany). Nuclear magnetic resonance (NMR) spectra
(1 H, 13 C and 2D-HSQC) of CQD samples dissolved in D2 O were recorded using a Bruker Avance
600 MHz spectrometer (Karlsruhe, Germany). Visual structures of each CQD sample was examined
by transmission electron microscopy (TEM) using a JEM 2100 transmission electron microscope
(JEOL Ltd., Tokyo, Japan). CQD photoluminescent spectra were recorded using FLS980 fluorescence
spectrophotometer (Edinburgh Instruments, Ltd., Livingston, UK) in aqueous solution. The quantum
yield of the CQD samples were determined at an excitation wavelength of 360 nm, using quinine
sulfate as the reference. X-ray photoelectron spectroscopy (XPS) spectra of CQD samples were also
acquired using an Ultima IV spectrometer (Rigaku, Japan).

2.4. Cell Viability Evaluation of the Synthesized CQDs

Cell viability of CQD-WS and CQD-BR were estimated using
a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in L929 fibroblasts. In the
first step of the assay, fibroblasts were seeded into 96-well plates, with 6000–7000 cells in 200 mL of
culture medium per well. The plates were then incubated at 37 ◦ C in a 5% CO2 atmosphere for 24 h
to allow the cells to attach to the wells. Next, 100 µL of CQD-WS and CQD-BR solution at different
concentrations (0, 25, 50, 100, 200 and 400 µg/mL) and 20 µL of MTT solution were added to each well
and further incubated at 37 ◦ C for 4 h. After incubation, the absorbance of each well were measured at
490 nm by a microplate reader. All assays were performed at least three times.

2.5. Cell Imaging of Synthesized CQDs

To begin the process of cell imaging, murine myeloma cells line SP2/0 were seeded into 6-well
culture plates containing 10% calf serum, 2.2 g/L NaHCO3 , 100 U/mL penicillin, and 100 g/mL
streptomycin in 5% CO2 at 37 ◦ C for 24 h. Next, 1 mL of the CQD solution at the concentration of
100 µg/mL (dissolved in phosphate buffered saline (PBS), pH = 7.4) was added in the plate and further
 Nanomaterials 2019, 9, 387 4 of 11

Nanomaterials 2019, 9, x FOR PEER REVIEW 4 of 11

incubated for 3 h. After incubation, the SP2/0 cells were washed with PBS solution three times to
remove
remove extracellular CQD.
extracellular After
CQD. washing,
After thethe
washing, coverslips were
coverslips placed
were on on
placed a glass microscope
a glass slide
microscope to to
slide
affix thethe
affix cellcell
andand
cellcell
fluorescence images
fluorescence were
images acquired
were using
acquired a confocal
using laser
a confocal scanning
laser microscope.
scanning microscope.

2.6.2.6.
In Vivo Bioimaging
In Vivo for for
Bioimaging Tumor CellCell
Tumor of Synthesized CQDs
of Synthesized CQDs
TheTheininvivo
vivo bioimaging abilityof of
bioimaging ability CQD-WS
CQD-WS and and
CQD-BRCQD-BR were examined
were examined using
using mouse mouse
experiments
experiments conducted according to the regulations of the He Nan Medical
conducted according to the regulations of the He Nan Medical Research center. Smmc-7721 tumorResearch center.
Smmc-7721
cells weretumor
used cells were
as the usedthat
tumor as the
wastumor that in
induced was
theinduced in the nude
nude mouse; 4 nmol mouse;
of the4 nmol
CQD of the
solution
CQD solution was injected into the nude tumor-bearing mouse by tail vein injection.
was injected into the nude tumor-bearing mouse by tail vein injection. In vivo fluorescence imaging In vivo
fluorescence
images (~5imaging
min–24 h)images
of the(~5
nudemin–24
mouse h)with
of the nudeand
tumor mouse
CQDwith tumor
injection andrecorded
were CQD injection were
by a NightOwl
recorded
LB 983 by a NightOwl
three LB small-animal
dimensional 983 three dimensional small-animal
imaging system. imaging with
After injection system. After solution
the CQD injectionfor
with the CQD solution for 24 h, the mouse was euthanized and its major tissue and
24 h, the mouse was euthanized and its major tissue and organs were dissected to obtain additionalorgans were
dissected to obtain
fluorescence additional fluorescence images.
images.

3. Results
3. Results andand Discussion
Discussion

3.1.3.1. Characterization
Characterization of As-Synthesized
of As-Synthesized CQDs
CQDs
TheThe green
green andand low-cost
low-cost precursor
precursor byproducts
byproducts in the
in the bio-refinery
bio-refinery process,
process, which
which areare obtained
obtained
from the autohydrolyzate in a biorefinery waste stream, are reacted in the
from the autohydrolyzate in a biorefinery waste stream, are reacted in the one-pot hydrothermal one-pot hydrothermal
treatment
treatment forfor
4 h4without
h without adding
adding anyany strong
strong acids,
acids, oxidizers,
oxidizers, andand metal.
metal. UreaUrea is used
is used as doped
as doped agentagent
to prepare
to prepare amino-passivated
amino-passivated CQDs CQDs fromfromthethe byproducts.
byproducts. TheThe facile
facile andand environmentally
environmentally friendly
friendly
treatment resulted in the as-synthesized CQDs possessing a uniform particle size that is less than88nm,
treatment resulted in the as-synthesized CQDs possessing a uniform particle size that is less than
nm,asas
shown
shown inin
thethe
TEM
TEM images
images in in
Figure
Figure1. 1.
Concerning
Concerning each sample,
each sample,CQD-WS
CQD-WS waswasrevealed
revealed to have
to
a narrow
have a narrowdistribution mostly
distribution in the
mostly insize
the range of ~1–6
size range ofnm.
~1–6Their
nm. average size wassize
Their average 3.5 was
nm (Figure
3.5 nm1c).
Separately,
(Figure CQD-BR also
1c). Separately, comprised
CQD-BR a uniformadistribution
also comprised but the particle
uniform distribution but the diameters
particle were smaller
diameters
~1–3
were nm (Figure
smaller ~1–3 1f).
nm High-resolution TEM shows that
(Figure 1f). High-resolution TEMmost of the
shows CQD-WS
that most ofand theCQD-BR
CQD-WSparticlesand
contained a well-resolved lattice spacing of 0.23 nm (Figure 1b inset) and 0.11
CQD-BR particles contained a well-resolved lattice spacing of 0.23 nm (Figure 1b inset) and 0.11 nm nm (Figure 1e inset),
(Figure 1e inset), respectively. From this analysis, it can be concluded that the CQD materialsthe
respectively. From this analysis, it can be concluded that the CQD materials synthesized from
biorefinery
synthesized pretreatment
from byproduct
the biorefinery are indeed
pretreatment the quantum-sized
byproduct are indeed the materials.
quantum-sized materials.

（a） （b） （c）

35
30
25
Pencentage(%)

20
15
10
5
0
2 3 4 5 6
Diameter(nm)

（d） （e） （f）

25

20
Pencentage(%)

15

10

0
1.0 1.5 2.0 2.5 3.0
Diameter(nm)

Figure
Figure 1. The
1. The transmission
transmission electron
electron microscope(TEM)
microscope (TEM)(a(a,d),
and high-resolution TEMTEM
d), high-resolution (HR-TEM) images
(HR-TEM)
(b,e) and particle size distributions (c,f) of CQD-WS and CQD-BR, respectively.
images (b and e) and particle size distributions (c and f) of CQD-WS and CQD-BR, respectively.

The functional groups on the surfaces of both CQD samples were investigated by comparing
the Fourier transform infrared (FT-IR) spectra obtained and shown in Figure 2a. A broad peak
Nanomaterials 2019, 9, 387 5 of 11

The functional groups on the surfaces of both CQD samples were investigated by comparing
Nanomaterials
the Fourier2019, 9, x FOR PEER
transform REVIEW
infrared (FT-IR) spectra obtained and shown in Figure 2a. A broad5peak of 11

centered at 3200 cm−1 corresponds to the stretching vibration and in-plane bending vibration of O-H
and N–H. Peaks at 1664 cm−1 and 1108 cm−1 are attributed to C=O and C–O stretching vibrations,
and N–H. Peaks at 1664 cm−1 and 1108 cm−1−1are attributed −1to C=O and C–O stretching vibrations,
respectively. The absorption band at 1580 cm and 1401 cm are assigned to the bending vibration
respectively. The absorption band at 1580 cm−1 and 1401 cm−1 are assigned to the bending vibration of
of C=C/N–H and C–N stretching. The stretching vibration and bending vibration of C–H were
C=C/N–H and C–N stretching. The stretching vibration and bending vibration of C–H were observed
observed at 3037 cm−1 and 1108 cm−1, respectively. All the information from the FT–IR spectra
at 3037 cm−1 and 1108 cm−1 , respectively. All the information from the FT–IR spectra provides proof
provides proof that hydroxyl, carboxyl, and amino groups are present at the surfaces of the CQDs [4,13].
that hydroxyl, carboxyl, and amino groups are present at the surfaces of the CQDs [4,13].
NMR spectroscopy (1H, 13C and 2D-Heteronuclear Single Quantum Coherence (2D-HSQC)) was
NMR spectroscopy (1 H, 13 C and 2D-Heteronuclear Single Quantum Coherence (2D-HSQC)) was
employed to distinguish the types of hybridized carbon atoms and discernible chemical structures
employed to distinguish the types of 13hybridized carbon atoms and discernible chemical structures
present in the prepared CQDs. In the C NMR spectra of CQD-WS and CQD-BR (Figure 2b), signals
present in the prepared CQDs. In the 13 C NMR spectra of CQD-WS and CQD-BR (Figure 2b), signals
were detected in the range of 15–70 ppm and 100–185 ppm. These signals correspond to both
were detected in the range of 15–70 ppm and 100–185 ppm. These signals correspond to both aliphatic
aliphatic carbon atoms (sp3) and sp2 hybridized carbon atoms, respectively. Signals between 170–185
carbon atoms (sp3 ) and sp2 hybridized carbon atoms, respectively. Signals between 170–185 ppm were
ppm were attributed to the carbon nuclei of carboxylic acid groups at the surface of the CQD [27]. In
attributed to the carbon nuclei of carboxylic acid groups at the surface of the CQD [27]. In the 1 H NMR
the H NMR spectra (Figure S1), sp hybridized carbon atoms signals were also found in the range of
1 2
spectra (Figure S1), sp2 hybridized carbon atoms signals were also found in the range of 7–9 ppm,
7–9 ppm, suggesting both CQD samples possess aromatic structures. This can be also confirmed
suggesting both CQD samples possess aromatic structures. This can be also confirmed from their
from their corresponding signals in 2D-HSQC spectra (Figure S2) [28,29], in which the aromatic
corresponding signals in 2D-HSQC spectra (Figure S2) [28,29], in which the aromatic structures signals
structures signals can be obviously observed in the region of δC/δH 100–135/5.5–8.5 ppm.
can be obviously observed in the region of δC /δH 100–135/5.5–8.5 ppm.
Surface groups and chemical structures were also investigated by X-ray photoelectron
Surface groups and chemical structures were also investigated by X-ray photoelectron
spectroscopy (XPS). The results from XPS spectra (Figure 2c) indicate that both CQD-WS and
spectroscopy (XPS). The results from XPS spectra (Figure 2c) indicate that both CQD-WS and CQD-BR
CQD-BR consist of carbon, nitrogen, and oxygen. This is provided by observation of the following
consist of carbon, nitrogen, and oxygen. This is provided by observation of the following peaks: C 1s,
peaks: C 1s, O 1s, and N 1s at 284.9 eV, 532.4 eV, and 399.25 eV, respectively. The C 1s XPS spectra
O 1s, and N 1s at 284.9 eV, 532.4 eV, and 399.25 eV, respectively. The C 1s XPS spectra (Figure 2d,e) reveal
(Figure 2d and 2e) reveal four peaks at 284.76 eV, 285.4 eV, 286.23 eV, and 288.6 eV for C–C/C=C,
four peaks at 284.76 eV, 285.4 eV, 286.23 eV, and 288.6 eV for C–C/C=C, C–N, C–O, and C=O/C=N,
C–N, C–O, and C=O/C=N, respectively [4]. All of the analytical results presented thus far indicate
respectively [4]. All of the analytical results presented thus far indicate that the prepared CQDs possess
that the prepared CQDs possess a naturally conjugated structure with passivated surfaces carrying
a naturally conjugated structure with passivated surfaces carrying aliphatic and aromatic hydroxyl
aliphatic and aromatic hydroxyl groups, carboxylic acid groups and amino groups. These structural
groups, carboxylic acid groups and amino groups. These structural features, in particular the surface
features, in particular the surface active amino groups, suggest that the as-prepared CQDs are
active amino groups, suggest that the as-prepared CQDs are suitable for luminescence emission.
suitable for luminescence emission.
(a) CQD-WS
(b)
CQD-BR
sp2C, C=O sp3C, C-C,C-O,C-N

616
3037 1580
1401 1108 767
3200 1664

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1 Chemical Shif (ppm)

(C)
(c) (d)
(d) (e)
(e)
C1s
O1s CQD-BR Raw Raw
CQD-WS C-C/C=C C-C/C=C
C-N C-N
C: 65.7%
O: 27.1% C-O C-O
Intensity (a.u)

C=O/C=N C=O/C=N
Intensity (a.u)

N: 7.2%
Intensity (a.u)

N1s

O1s
C: 69.5%
C1s
O: 26.4%
N1s N: 4.1%

200 400 600 800 1000 1200 282 284 286 288 290 282 284 286 288 290

Binding Energy (eV) Binding Energy (eV) Binding Energy (eV)

Figure 2. (a) Fourier transform–infrared (FT–IR) spectra, (b) 13 C nuclear magnetic resonance (NMR)
Figure 2. (a) Fourier transform–infrared (FT–IR) spectra, (b) 13C nuclear magnetic resonance (NMR)
spectra, (c) X-ray photoelectron spectroscopy (XPS) survey spectra of CQD-WS and CQD-BR, (d) C 1s
spectra, (c) X-ray photoelectron spectroscopy (XPS) survey spectra of CQD-WS and CQD-BR, (d) C
XPS spectra of CQD-WS and (e) C 1s XPS spectra of CQD-BR.
1s XPS spectra of CQD-WS and (e) C 1s XPS spectra of CQD-BR.

3.2. Photophysical Properties of As-Synthesized CQDs

Nanomaterials 2019, 9, 387 6 of 11
Nanomaterials 2019, 9, x FOR PEER REVIEW 6 of 11

The optical properties

3.2. Photophysical Properties ofofAs-Synthesized
each CQD were CQDscharacterized to further investigate these material’s
suitability for bioimaging. As shown in Figure 3a,b,
The optical properties of each CQD were characterized the ultraviolet–visible (UV–Vis) these
to further investigate spectra for both
material’s
CQD-WS and CQD-BR contain an absorbance peak at 280 nm. This peak is
suitability for bioimaging. As shown in Figure 3a,b, the ultraviolet–visible (UV–Vis) spectra attributed to the π–π*
for
transition of the aromatic carbon bonds. In the Figure 3c,d, it can be seen
both CQD-WS and CQD-BR contain an absorbance peak at 280 nm. This peak is attributed to the that the fluorescence
intensities of CQD-WS
π–π* transition and CQD-BR
of the aromatic carbondependent
bonds. In theon Figure
the fluorescence
3c,d, it can emission.
be seen that Visually, it can be
the fluorescence
seen
intensities of CQD-WS and CQD-BR dependent on the fluorescence emission. Visually, it can be of
that the prepared CQDs are easily dispersible in water and have the appearance seena
transparent, uniform,
that the prepared CQDs andare
yellow
easilysolution under
dispersible normal
in water light.
and have However, they exhibit
the appearance blue-green
of a transparent,
fluorescence
uniform, and yellow solution under normal light. However, they exhibit blue-green fluorescencein
under 365 nm UV light irradiation (Fig 3a and Fig 3b inset). With quinine sulfate 0.10
under
M
365Hnm
2SO4 as the reference, the synthesized CQDs were measured with quantum yield of ~13% under
UV light irradiation (Figure 3a,b inset). With quinine sulfate in 0.10 M H2 SO4 as the reference,
the excitation
the synthesized of CQDs
365 nm, which
were is a relatively
measured high degree
with quantum and
yield of further proves
~13% under the
the material’s
excitation ofviability
365 nm,
in bioimaging.
which is a relatively high degree and further proves the material’s viability in bioimaging.

(a) (b) EX
EX EM EM

PL intensity (a.u)
PL intensity (a.u)

Abs
Absorbance
Abs

Absorbance
200 300 400 500 600 700 200 300 400 500 600 700
Wavelength (nm) Wavelength (nm)
(c) 360 nm (d) 360 nm
370 nm 370 nm
380 nm 380 nm
PL intensity (a.u)

390 nm 390 nm
PL intensity (a.u)

400 nm
400 nm
410 nm 410 nm
420 nm
420 nm
430 nm 430 nm
440 nm

400 450 500 550 600 650 700 400 450 500 550 600 650 700
Wavelength (nm) Wavelength (nm)

Figure 3.
Figure Ultraviolet–visible (UV–Vis),
3. Ultraviolet–visible (UV–Vis),fluorescence
fluorescenceexcitation
excitationand
andemission
emissionspectra of of
spectra thethe
CQD-WS (a)
CQD-WS
and CQD-BR (b); excitation-dependent fluorescence emission of CQD-WS (c) and CQD-BR (d).
(a) and CQD-BR (b); excitation-dependent fluorescence emission of CQD-WS (c) and CQD-BR (d).

The fluorescence emission intensity of the surface state/molecule state of the CQDs is strongly
The fluorescence emission intensity of the surface state/molecule state of the CQDs is strongly
affected by surrounding factors like environmental temperature and UV irradiation. Figure S3 shows
affected by surrounding factors like environmental temperature and UV irradiation. Figure S3
the fluorescent behavior of the CQD-WS and CQD-BR observed at 4–60 ◦ C. It can be seen that the
shows the fluorescent behavior of the CQD-WS and CQD-BR observed at 4–60 °C. It can be seen that
temperature showed low effects on the CQD fluorescence properties. Importantly, it is found that
the temperature showed low effects on the CQD fluorescence properties. Importantly, it is found
there were no obvious changes in fluorescent intensity or peak excursion at different temperatures.
that there were no obvious changes in fluorescent intensity or peak excursion at different
When they were irradiated by UV at 365 nm, just a slight decrease in fluorescence intensity (7% and
temperatures. When they were irradiated by UV at 365 nm, just a slight decrease in fluorescence
9%, Figure 4 and Figure S4) occurred after the they were photobleached for 90 min. This decrease was
intensity (7% and 9%,0 Figure 4 and Figure S4) occurred after the they were photobleached for 90
minor compared to 4 ,6-diamidino-2-phenylindole (DAPI), a commercial fluorescent display agent
min. This decrease was minor compared to 4′,6-diamidino-2-phenylindole (DAPI), a commercial
currently used in bioimaging. For DAPI, an obvious decrease in fluorescence intensity (about ~45–50%)
fluorescent display agent currently used in bioimaging. For DAPI, an obvious decrease in
was found after similar photobleaching treatment [29]. This different indicates that the prepared CQDs
fluorescence intensity (about ~45–50%) was found after similar photobleaching treatment [29]. This
are more durable and robust compared to the commercial material. Hence, it is speculated that the
different indicates that the prepared CQDs are more durable and robust compared to the
CQDs synthesized from a biorefinery liquid byproduct can provide a sustainable alternative chemical
commercial material. Hence, it is speculated that the CQDs synthesized from a biorefinery liquid
in the field of bioimaging.
byproduct can provide a sustainable alternative chemical in the field of bioimaging.
 Nanomaterials
Nanomaterials 2019,
2019, 9,
9, x387
FOR PEER REVIEW 77of
of 11
11
Nanomaterials 2019, 9, x FOR PEER REVIEW 7 of 11
0.4
0.4

0.2

0/I 0)
0.2

0/I 0)
(I-I(I-I
0.0

intensity
0.0

intensity
-0.2

PL PL
CQD-WS
-0.2 CQD-BR
CQD-WS
CQD-BR
-0.4
-0.40 10 20 30 40 50 60 70 80 90
0 10 20 30 40 50Time
Photobleaching 60 (min)
70 80 90
Photobleaching Time (min)
Figure 4. Fluorescence intensity of CQD-WS and CQD-BR upon irradiation with UV light at 365 nm
Figure
Figure4. Fluorescence intensity of
ofCQD-WS
CQD-WSand
andCQD-BR
CQD-BR upon
upon irradiation
irradiation with
with UV
UV light
light at
at 365
365nm
(0.1 mW4.cmFluorescence
−2) in aqueousintensity
solution. nm
(0.1 mW cm −2 ) in aqueous solution.
(0.1 mW cm−2) in aqueous solution.
3.3.
3.3. Biocompatibility Evaluation
Biocompatibility Evaluation of As-Synthesized
of As-Synthesized CQDsCQDs by Usingby Using Fibroblasts
Fibroblasts and Murine andMyeloma
Murine Cells
3.3. Biocompatibility
Myeloma Cells Evaluation of As-Synthesized CQDs by Using Fibroblasts and Murine
Biocompatibility
Myeloma Cells and bioviability are a crucial factors that must be considered when investigating
Biocompatibility
carbon quantum dot nanoparticlesand bioviability are a crucial
for the bioimaging factors
of a cell thatTomust
[30,31]. evaluate be the
considered when
biocompatibility
Biocompatibility
investigating
behavior of the carbon
prepared and
quantum
CQDs,bioviability
dot are
wenanoparticles a
examined thefor crucial
inthe factors
bioimaging
vitro that
cytotoxicity of ofmust
a cell be
the [30,31]. considered
samplesTo evaluate
using when
the
an assay
investigating
biocompatibility carbon quantum
behavior of dot
the nanoparticles
prepared CQDs, for the
we bioimaging
examined of
the a
in
involving L929 fibroblasts. As shown in Figure 5a, the cell viability remained greater than 90% whencell
vitro[30,31]. To evaluate
cytotoxicity of the
the
biocompatibility
samples
the concentrations behavior
using an assay
of both of theL929
involving
CQD-WS prepared
and CQD-BR CQDs,
fibroblasts. As we
shown
increased examined
in 25
from to the
Figure 4005a,inthevitro
mg/mL. cytotoxicity
cellThis
viability of the
remained
demonstration
samples
greater
indicates using
than
that90%an assay
when
they have involving
theexcellent L929
concentrations fibroblasts. As
of both CQD-WS
biocompatibility, shown
providing in Figure
and further
CQD-BR 5a, the cell
increased
proof viability remained
from materials
that these 25 to 400
greater
mg/mL. than
This 90% when
demonstration the concentrations
indicates that of both
they haveCQD-WS
excellent and
are suitable for bioimaging. The results are in accordance with the biocompatibility of CQD-BR
biocompatibility,increased from
providing 25further
to 400
CQDs
mg/mL.
proof thatThis demonstration
these materials areindicates
suitable that
for they have
bioimaging. excellent
The biocompatibility,
results are
obtained from different sugars and biomass [4,13]. To explore the biological properties of the CQDs, in providing
accordance withfurther
the
proof that
biocompatibility these materials
of CQDs obtained
2,2-diphenyl-1-picryl-hydrazyl are suitable
(DPPH)from for
radical bioimaging.
different
scavenging sugars The
assaysandresults
were are
biomass
performed in accordance
[4,13]. To explore
to evaluate with
whether the
the
biocompatibility
biological
the samples properties of of
also have CQDs
any obtained
theadditionally
CQDs, from different sugarscapacity.
2,2-diphenyl-1-picryl-hydrazyl
beneficial antioxidant and(DPPH)
biomass
From [4,13].
radical
Figure Toit explore
scavenging
5b, can be the
assays
seen
biological
were
that both properties
performed
CQD-WS of the
to evaluate
and CQD-BR CQDs,
whether 2,2-diphenyl-1-picryl-hydrazyl
the samples
exhibited also have
scavenging any on
activities (DPPH)
additionally radical
DPPH radicals scavenging
beneficial assays
antioxidant
in vitro, with the
were
capacity.performed
From to
Figureevaluate
5b, it whether
can
maximum scavenging ability ~40% at 0.6 mg/mL. be the
seen samples
that both also have
CQD-WS anyandadditionally
CQD-BR beneficial
exhibited antioxidant
scavenging
capacity.on
activities From
DPPH Figure 5b, in
radicals it vitro,
can bewithseenthethat both CQD-WS
maximum scavenging andability
CQD-BR ~40%exhibited scavenging
at 0.6 mg/mL.
120 activities on DPPH radicals in vitro, with the maximum (b) 60scavenging ability ~40% at 0.6 mg/mL.
(a)
120 CQD-WS
(a) CQD-BR
(b) 60
100 100 CQD-WS 50
% %

100 100 CQD-BR

50
(% )(% )

Ability

80 80 40
Ability
Viability

80 80 40
60
Viability

60
30
Scavenging

60 60 30
Scavenging

40 40 20
CellCell

40 40 20 CQD-WS
20 20 10 CQD-BR
CQD-WS
20 20 10 CQD-BR
0 0 0
0 25 50
0 0100 200 400 0.2 0.4 0.6 0.8 1.00.0
0
25 0
50 (mg/mL)
Concentration 100 200 400 Concentration
0.2 0.4 (mg/mL)
0.6 0.8 1.00.0
Concentration (mg/mL) Concentration (mg/mL)
Figure 5.
Figure 5. (a)
(a)Cell
Cellviability
viabilityof L929 fibroblasts
of L929 after incubation
fibroblasts with different
after incubation concentrations
with different of CQD-WS
concentrations of
and CQD-BR
Figure 5. and
CQD-WS for 24
(a) Cell h;
CQD-BR (b) 2,2-diphenyl-1-picryl-hydrazyl
viability of L929
for 24 h; (b)fibroblasts (DPPH) radical scavenging
after incubation with(DPPH)
2,2-diphenyl-1-picryl-hydrazyl different ability of CQD-WS
concentrations
radical scavenging of
and CQD-BR.
CQD-WS
ability and CQD-BR
of CQD-WS for 24 h; (b) 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging
and CQD-BR.
ability of CQD-WS and CQD-BR.
Based on the good biocompatibility, CQD-WS and CQD-BR were introduced into SP2/0 murine
Based on the good biocompatibility, CQD-WS and CQD-BR were introduced into SP2/0 murine
myeloma cells
Basedcells
on theline for in vitro bioimaging by confocal laser scanning microscopy. As shown in
myeloma linegood
for inbiocompatibility,
vitro bioimaging CQD-WS and CQD-BR
by confocal were introduced
laser scanning intoAs
microscopy. SP2/0 murine
shown in
Figure
myeloma6a,d, the profile of SP2/0 is only slightly observable in the bright-field. However, the cells
Figure 6a,d,cells
the line for of
profile in SP2/0
vitro bioimaging
is only slightlyby confocal
observablelaser scanning
in the microscopy.
bright-field. As shown
However, the cells in
exhibited
Figure thethe
6a,d, intended
profile blue
of fluorescence
SP2/0 is only and red
slightly fluorescence
observable in upon
the incubation
bright-field. with CQD-WS
However, the cells
exhibited the intended blue fluorescence and red fluorescence upon incubation with CQD-WS
(Figure 6b,e)
exhibited and
theand CQD-BR
intended blue(Figure 6c,f) at excitation
fluorescence wavelengths upon
and red wavelengths
fluorescence of 420 and 540 nm, respectively.
(Figure 6b,e) CQD-BR (Figure 6c,f) at excitation of 420 incubation
and 540 nm,with CQD-WS
respectively.
(Figure 6b,e) and CQD-BR (Figure 6c,f) at excitation wavelengths of 420 and 540 nm, respectively.
Nanomaterials 2019, 9, 387 8 of 11

These results indicated that the synthesized CQDs from byproducts in the bio-refinery process can
Nanomaterials 2019, 9, x FOR PEER REVIEW 8 of 11
indeed function as bioimaging agents.

(a) (b) (c)

50 祄 50 祄 50 祄

(d) (e) (f)

100 祄 100 祄 100 祄

Figure 6. Confocal laser scanning microscopy images of SP2/0 murine myeloma cells at bright-field
(a,d),Figure 6. Confocal
fluorescence laser
images scanning
of the microscopy
cells incubated images
with of SP2/0
CQD-WS murine
(b,e) and myeloma
CQD-BR (c,f) cells at bright-field
solution under
(a and d), fluorescence images of the cells incubated
excitation wavelengths of 420 and 540 nm, respectively. with CQD-WS (b and e) and CQD-BR (c and f)
solution under excitation wavelengths of 420 and 540 nm, respectively.
3.4. Bioimaging Applications for Tumor Cell of As-Synthesized CQDs
3.4.
ForBioimaging
the in vivoApplications for Tumor
optical bioimaging Cell of aAs-Synthesized
studies, nude mouse wasCQDs
inoculated with Smmc-7721 tumor
cells. TheFor the imaging
optical in vivo ofoptical bioimaging
the CQD-WS studies, a nude
was investigated mouse was
by intravenous inoculated
injection of 200with Smmc-7721
uL CQD-WS
tumor cells.
(0.2ug/mL) intoThethe optical
mouseimaging of the
via the tail CQD-WS
vein. Opticalwas investigated
images by intravenous
of distribution injectionin
of the CQD-WS of the
200 uL
CQD-WS (0.2ug/mL)
tumor-bearing mouse over into the mouse
increasing via the
times are tail vein.inOptical
shown Figureimages of distribution
7a. In addition, optical ofimages
the CQD-WSof
in the tumor-bearing
fluorescence intensities withinmouse the over increasing
harvested organstimes are shown
are shown in Figurein Figure 7a. Inthe
7b. Without addition,
injectionoptical
of
imagesno
CQD-WS, offluorescent
fluorescence intensities
signals within the
were detected in harvested organs are
the tumor-bearing shownbody
mouse’s in Figure
(Figure 7b.7a,Without
0 min). the
5 mininjection of CQD-WS,
after injecting no fluorescent
the CQD-WS solution, signals
fluorescencewere signals
detected in the tumor-bearing
gradually became detectable, mouse’s
first atbody
(Figure
the head 7a, 0mouse.
of the min). 5Asmin after
time injecting the
progressed CQD-WS
towards solution,
3 h, the CQD-WS fluorescence
circulatedsignals
in the gradually
mouse’s body became
and detectable, first at the head
gradually congregated at theof location
the mouse. As time
of the tumor.progressed towardsindicates
This observation 3 h, the CQD-WS circulated in
that the CQD-WS
can the mouse’s body and gradually congregated at the location of the tumor. This observation indicates
cycle in a mouse’s body and also aggregate where it is intended to. After 12 h, it was found that
that the
CQD-WS CQD-WSalmost
stabilized can cycle in a mouse’s
exclusively at thebody and also
location of theaggregate
tumor. Thiswhere is it is intended
another to. After 12 h,
demonstration
thatitthe
was found
CQD hasthat
goodCQD-WS stabilized almost
biocompatibility and could exclusively
be usedat forthe location
tumor of the tumor.
bioimaging over This is another
prolonged
demonstration
periods of time. Figurethat7bthe
showsCQD hasthe
that good biocompatibility
dissected kidney, liver,and
and could
tumorbe fromused
the for tumor bioimaging
euthanized mouse
over fluorescence,
showed prolonged periods of time.
revealing thatFigure 7b showswas
the CQD-WS thattaken
the dissected
up by thesekidney, liver,
organs and tumor
[32,33]. from the
However,
euthanized mouse showed fluorescence, revealing that the CQD-WS was taken up by these organs
no fluorescence signals were detected in the organs of the heart, lung, and spleen. Overall, most of
[32,33]. However,
the CQD-WS aggregated no fluorescence
in the tumorsignals were detected
and fluoresced in theindicating
intensely, organs of thatthe heart, lung, and spleen.
the as-synthesized
Overall,
CQD-WS wasmost of thebioimaging
a viable CQD-WS aggregated
agent and in thehas
thus tumor and fluoresced
immense potential intensely,
in relatedindicating
fields suchthat as the
as-synthesized CQD-WS was a viable bioimaging agent and thus has immense potential in related
phototherapy and bioimaging.
fields such as phototherapy and bioimaging.
Nanomaterials 2019, 9, 387 9 of 11
Nanomaterials 2019, 9, x FOR PEER REVIEW 9 of 11

(a)

0min 5 min 30 min 1h 2h

3h 4h 8h 12 h 24 h

(b)

Heart Lung Spleen Kidney Liver Tumor

Figure 7. (a) In vivo fluorescence imaging of nude mice after intravenous injection of CQD-WS solution;
Figure 7. (a) In vivo fluorescence imaging of nude mice after intravenous injection of CQD-WS
(b) representative fluorescence images of dissected organs of a mouse after intravenous injection of
solution; (b) representative fluorescence images of dissected organs of a mouse after intravenous
CQD-WS solution for 24 h.
injection of CQD-WS solution for 24 h.
4. Conclusions
4. Conclusions
CQDs with narrow size distribution from 2–6 nm were successfully synthesized using a one-pot
CQDs withreaction
hydrothermal narrow using
size distribution from
a biorefinery 2–6 nm were
byproduct successfully synthesized
(autohydrolyzate) doped with using a one-pot
urea. NMR,
hydrothermal
FT–IR, and XPS spectroscopy confirmed the associated chemical moieties in the CQDs hadNMR,
reaction using a biorefinery byproduct (autohydrolyzate) doped with urea. high
FT–IR, and XPS spectroscopy
2 3 confirmed the associated chemical moieties in the
percentages of sp and sp groups. The CQDs showed stable fluorescence behavior, even under CQDs had high
percentages of sp2 and and
changing temperatures sp3 groups.
undergoingThephotobleaching
CQDs showed treatment.
stable fluorescence behavior,
In addition, evenimaging
in vivo cell under
changing temperatures and undergoing photobleaching treatment. In addition, in
and biomaging experiments showed that the CQDs have low cytotoxicity, antioxidant activity, vivo cell imaging
and
and biomaging
an overall experiments showed that
excellent bioimaging the CQDs have
performance. low
In all, cytotoxicity,
our antioxidant
work demonstrates activity,
a novel and
means
an overall excellent bioimaging performance. In all, our work demonstrates a novel
of producing high-value materials that can be applied in advanced bioimaging applications using only means of
producing high-value
a liquid byproduct materials
from that processes.
biorefinrey can be applied in advanced bioimaging applications using only
a liquid byproduct from biorefinrey processes.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/9/3/387/s1,
Supplementary
Figure S1: 1 H NMR Materials:
spectraThe followingand
of CQD-WS areCQD-BR,
availableFigure
online S2:
at www.mdpi.com/xxx/s1,
2D-HSQC spectra of CQD-WSFigure and
S1: 1H NMR
CQD-BR,
Figure S3: the fluorescent behavior of the CQD-WS and CQD-BR observed at 4–60 ◦ C, Figure S4: Fluorescence
spectra of CQD-WS and CQD-BR, Figure S2: 2D-HSQC spectra of CQD-WS and CQD-BR, Figure S3: the
intensity of CQD-WS and CQD-BR upon irradiation with UV light at 365 nm (0.1 mW cm−2 ) in aqueous solution.
fluorescent behavior of the CQD-WS and CQD-BR observed at 4–60 °C, Figure S4: Fluorescence intensity of
CQD-WS and CQD-BR upon
Author Contributions: irradiation
Data curation, withY.S.
H.D., UVand
lightY.W.;
at 365 nm (0.1 mWH.D.,
Investigation, cm−2)Y.S.
in aqueous
and Y.W.;solution.
Supervision, Q.Y.;
Writing—original draft, C.H.; Writing—review and editing, R.N.
Author Contributions: Data curation, Huiling Dong, Yan Su and Yan Wu; Investigation, Huiling Dong, Yan Su
Funding: This work was supported by the Natural Science Foundation of Jiangsu Province (BK20180772) and the
and Yan Wu;
National Supervision,
Natural Qiang Yong;
Science Foundation of Writing―original
China (31800501). draft, Caoxing Huang; Writing―review and editing,
Robert Narron.
Acknowledgments: We thank Shilong Yang in the Advanced Analysis and Testing Center of Nanjing Forestry
Funding:
UniversityThis workwith
for help wasthe
supported
work. by the Natural Science Foundation of Jiangsu Province (BK20180772) and
the National Natural Science Foundation
Conflicts of Interest: The authors declareofnoChina (31800501).
conflict of interest.
Acknowledgments: We thank Shilong Yang in the Advanced Analysis and Testing Center of Nanjing Forestry
University
References for help with the work.

Conflicts
1. of Interest:
Essner, The
J.B.; Laber, authors
C.H.; declare
Ravula, no conflict ofL.;interest.
S.; Polo-Parada, Baker, G.A. Pee-dots: Biocompatible fluorescent carbon
dots derived from the upcycling of urine. Green Chem. 2016, 18, 243–250. [CrossRef]
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