Mg/N double doping strategy to fabricate extremely high luminescent carbon dots for bioimaging

Article abstract of DOI:10.1039/c3ra43826k

Carbon dots (CDs) with quantum yield up to 83% have been synthesized with a Mg/N double doping fluorescence-enhanced strategy. Besides N-mediated surface passivation by ethylenediamine, the Mg-citric acid chelate played the roles of introducing Mg and preserving the carboxyl group, both greatly contributing to the photoluminescence enhancement of the final CDs. Importantly, the N- and Mg-doping functioned in concert without mutual influence.

Full text of DOI:10.1039/c3ra43826k

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Mg/N double doping strategy to fabricate
extremely high luminescent carbon dots for
Cite this: RSC Adv., 2014, 4, 3201
bioimaging†
a
a
a
a
b
a
Fan Li, Changjun Liu,* Jian Yang, Zheng Wang, Wenguang Liu and Feng Tian*
Carbon dots (CDs) with quantum yield up to 83% have been synthesized with a Mg/N double doping
fluorescence-enhanced strategy. Besides N-mediated surface passivation by ethylenediamine, the Mg–
citric acid chelate played the roles of introducing Mg and preserving the carboxyl group, both greatly
contributing to the photoluminescence enhancement of the final CDs. Importantly, the N- and Mg-
doping functioned in concert without mutual influence.
Received 24th July 2013
Accepted 22nd October 2013
DOI: 10.1039/c3ra43826k
www.rsc.org/advances
Citric acid, a weak acid, has been reported to be used as carbon
source to prepare CDs in which the surface passivation agent
The surface-passivated carbonaceous quantum dots, so-called containing amino groups is necessary for yielding high PL.
Introduction
19,20
carbon dots (CDs), were discovered serendipitously by Herein, we explore a simple route to prepare highly uorescent
researchers purifying single-walled carbon nanotubes fabricated CDs using citric acid as carbon source even without resorting to
1
by arc-discharge methods. Since then, CDs have been attracting specialized surface passivation. It is well known that the metallic
considerable attention due to their versatile preparation routes, chelates possess preeminent thermal stability for their chelate
ready large scalability, better biocompatibility colloidal stability rings similar to aromatic rings, and it has been proved that some
2
–4
22
and particularly tunable photoluminescence (PL). In the last functional groups such as amino and carboxyl groups could be
decade, a variety of approaches have been explored for fabri- protected by chelating effect in the chemical reaction procedure.
cating CDs, which can be generally classied into two main So the chelation of citric acid by divalent metal ions can protect
5
groups: top-down and bottom-up methods. It has been the carboxyl groups of citric acid from over-consuming during the
conrmed by present reports that CDs can be produced inex- process of dehydration and carbonization. In this study, to prove
pensively on a large scale for future practical applications our hypothesis, we chose magnesium hydroxide (Mg(OH)
2
) as a
through the “bottom-up” methods such as combustion/ representative chelation agent. To produce much higher PL CDs,
6
–10
11–15
thermal or microwave methods.
CDs by strong acid or alkali was necessary to gain a strong uo- double doping strategy, will be attempted.
However, the treatment of Mg chelation in combination with nitrogen passivation, we called
16,17
rescent property in the early research.
Recently, some suitable
molecular precursors with mild surface passivators or even
without passivators are chosen to synthesize enhanced photo-
Experimental section
9,18–20
luminescent CDs using a one-step pathway.
In fact, all the Chemicals and reagents
CDs obtained from the “bottom-up” methods can be regarded as
the products of incomplete carbonization of carbohydrate.
Though some researchers have yet found the particle size of CDs
is closely related to their uorescence property, it is now widely
recognized that the surface state of CDs plays the key role in
enhancing its uorescence. So how to hold and enrich as much
as possible specic chemical groups on the surface of CDs may
be a facile way to enhance the PL.
Citric acid (99%) and 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl
tetrazolium bromide (MTT, 98%) were purchased from Alfa
Aesar. Ethylenediamine (98%) and magnesium hydroxide (98%)
were obtained from GuangFu Technology Development Co. Ltd.
Quinine sulfate (98%, suitable for uorescence) was supplied by
Fluka. All other reagents were of analytical grades and used
without further purication.
21
Preparation of high luminescent carbon dots
a
Institute of Medical Equipment, Academy of Military Medical Sciences, Tianjin
Typically, 10 g citric acid was dissolved in 40 mL distilled water
followed by an adequate addition of 4.2 g Mg(OH) in succes-
3
00161, PR China. E-mail: hunterlcj@163.com; tianfeng62037@163.com
b
2
School of Materials Science and Engineering, Tianjin Key Laboratory of Composite
and Functional Materials, Tianjin University, Tianjin 300072, PR China
sion to form a colorless, transparent and homogeneous solu-
tion without other anions. Then a hydrothermal procedure was
†
Electronic supplementary information (ESI) available: Experimental details and
ꢀ
supplementary data. See DOI: 10.1039/c3ra43826k
applied at 200 C for 3 h. The raw products were treated with
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further ltration, dialysis and lyophilization to obtain CDs measured on a Nicolet 380 spectrometer (Thermo, USA). The
denoted as Mg–CDs. Simultaneously, to gain a better under- surface states and composition of the samples were measured
standing of the role of magnesium chelator in the formation of on a Kratos AXIS Ultra DLD X-ray Photoelectron Spectroscopy
high uorescent CDs, parallel experiments were also carried out (XPS, Shimadzu, Japan). X-Ray diffraction (XRD) proles of the
as follows: (1) pyrolysis of citric acid without any doping, (2) prepared samples were recorded on a Rigaku-D/MAX 2500
using ethylenediamine (EDA) as surface passivation reagent diffractometer (Rigaku, Japan) equipped with graphite mono-
without addition of Mg(OH)
2
, and (3) with addition of both EDA chromatized CuKa (l ¼ 0.15405 nm) radiation at a scanning
ꢀ
ꢁ1
ꢀ
ꢀ
and Mg(OH) . The corresponding resultant products were speed of 4 min in the range from 5 to 60 .
2
denoted as original CDs, EDA–CDs and Mg–EDA–CDs, respec-
tively. More details were listed in the Table S1.†
Results and discussion
As one of the most important function indices, the quantum
yield (QY) of CDs was measured at 360 nm excitation wave-
length using quinine sulphate as a standard (QY ¼ 54%).
Remarkably, Mg–CDs exhibited a high QY value of 18.2%
Cell labeling and cytotoxicity assay
L929 cells were obtained from Peking Union Medical College
(Beijing, China). The cells were cultured in medium of RPMI
1
1
640 (1640, HyClone), containing 10% fetal bovine serum (FBS),
00 U mL penicillin and 100 mg mL streptomycin at 37 C
(
Fig. S1†); while the QY of CDs without any doping was merely
.8%. Amazingly, the QY of EDA–CDs reached 73.1%, which is
close to the latest published results nished by Yang and his
ꢁ1
ꢁ1
ꢀ
0
in 5% CO humidied atmosphere.
2
For confocal microscope, L929 cells were seeded on a glass
bottom culture dish (MatTek, USA) 12 h before use. Then the
culture medium was replaced by 2.5 mL fresh medium contain-
20
coworkers recently. Importantly, the QY of Mg–EDA–CDs was
further enhanced to as high as 83.0%, which is the highest value
for CDs reported so far and almost equal to that of organic dyes
or semiconductor quantum dots. Because of extremely high PL,
blue uorescent emission from aqueous solution of Mg–EDA–
CDs could be easily observed under a UV lamp or even under a
natural light (Fig. 1a).
ꢁ1
ing 100 mg mL CDs and the cells were incubated for another 24
h. The cells were washed with isotonic PBS (pH 7.4) three times.
Then the L929 cells were xed with 4% paraformaldehyde solu-
ꢀ
tion in PBS at 4 C overnight. The samples were examined under
a
Leica confocal laser scanning microscope (Mannheim,
The UV-Vis spectra of both EDA–CDs and Mg–EDA–CDs
Germany) equipped with a semiconductor laser (405 nm), an Ar
laser (457/488/514 nm) and a HeNe laser (543/633 nm).
The cytotoxicity of CDs was assessed through MTT assay. L929
(Fig. S2†) showed obvious and similar absorption peaks centred
at 346 nm ascribing to the n–p* transition, indicating the
existence of aromatic structures in the CDs. As to Mg–EDA–CDs,
a maximum PL emission peak was observed at around 437 nm
when excited at 360 nm, closely near the position of UV-Vis
absorption peak (Fig. 1a). However, we found a specic transi-
tion from primordial CDs to Mg–EDA–CDs in the PL emission
spectra (Fig. 1b, lex ¼ 360 nm). The non-doped CDs showed a
weaker peak at 459 nm compared with that of Mg–CDs at
4
cells were seeded in a 96-well plate, at a density of 2 ꢂ 10 cells per
well and incubated overnight. The following morning, the culture
medium in each well was replaced by 180 mL fresh RPMI 1640/FBS.
CDs solutions with various concentrations were then added to
each well. Aer incubation for 24 h, the medium containing CDs
was removed, and replaced with 200 mL fresh medium containing
ꢁ1
2
0 mL MTT (5 mg mL in PBS) and incubated for another 4 h.
437 nm. Likewise, EDA–CDs exhibited an emission peak at 449
Finally all medium was removed and 150 mL per well dimethyl
sulphoxide (DMSO) was added, followed by shaking for 15 min.
The absorbance of each well at 490 nm was measured using a
Synergy HT Multi-Mode Microplate Reader (BioTek, USA) with
pure DMSO as a blank. Non-treated cells (in RPMI 1640) were used
as a control and the relative cell viability (mean% ꢃ SD, n ¼ 3) was
expressed as Abssample/Abscontrol ꢂ 100%.
nm in contrast with a 437 nm peak of Mg–EDA–CDs. The results
revealed that an unknowable blue-shi phenomenon occurred
to the CDs once doped with magnesium.
To further explore optical properties of the as-prepared CDs,
we carried out a detailed PL study by changing excitation
wavelengths ranging from 360 to 500 nm (Fig. 1c). With the
increase of the excitation wavelength, the maximum emission
peak position shied to longer wavelength gradually but in a
non-uniform mode, simultaneously accompanied with
Measurements
UV-Vis absorption was measured on a TU-1810 UV-Vis spectro- remarkable decrease of PL intensity. As different energy levels
photometer (Pgeneral, China). PL emission measurements were associated with different “surface states” formed by different
performed using FLS920 uorometer (Edinburgh Instruments, functional groups are responsible for the excitation-dependent-
23
Britain). The normalized spectrum was obtained by divided emission phenomenon, the observed excitation-dependent
each intensity of the PL spectrum by the maximum value of its emission over 360–500 nm might be attributed to the rela-
own. The morphology and microstructure of the CDs were tively well-passivated but non-uniform CDs surface. The bath-
examined by high-resolution transmission electron microscopy ochromic shi phenomenon of emission from CDs has also
5,9,14,17
(HRTEM) on a Philips Tecnai G2 F20 microscope (Philips, been reported previously.
Netherlands) with an accelerating voltage of 200 kV. The
Additionally, the inuence of pH value on the PL intensity of
samples for HRTEM were made by dropping an aqueous solu- the as-prepared CDs was also investigated (Fig. S8†). The PL
tion onto a 300-mesh copper grid coated with a lacy carbon lm. intensity was very stable over the pH range of 3–11, which was
The Fourier transform infrared (FTIR) spectra of CDs were favorable for CDs applications in the physiological and
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Fig. 1 (a) UV-Vis and PL spectra (lex ¼ 360 nm) of the Mg–EDA–CDs in aqueous solution (insets: photographs under natural light and UV light).
(
b) PL spectra (lex ¼ 360 nm) of CDs with different doping strategies. (c) PL emission spectra of Mg–EDA–CDs (insets of (b) and (c): the
normalized PL emission spectra). (d) HRTEM image of Mg–EDA–CDs (scale bar: 20 nm).
pathological environments with a normal pH range of 4.5–9.5. successful anchoring of amino groups. Moreover, it is worth-
And the PL intensity decreased slightly when the pH value was 1 while to note that both Mg–CDs and Mg–EDA–CDs possess a
ꢁ
1
or 13. This change was associated with the surface chemical similar and prominent peak at 1400 cm ascribed to carboxy-
20
ꢁ
groups of the CDs.
late anion (–COO ). Further characterization studies using XPS
HRTEM, XRD and FTIR spectra were conducted to charac-
terize the micromorphology and chemical structure of CDs. The
HRTEM images, as shown in Fig. 1d, displayed that Mg–EDA–
CDs were well distributed among a narrow range between 0.8
and 2.8 nm without apparent agglomeration. From the magni-

ed images (Fig. S3†), we can see, the overwhelming majority of
CDs exhibited an amorphous structure; only a handful of CDs
appeared barely distinguishable lattice fringes in the core
24
area. Owing to such a low carbon-lattice-structure content, the
XRD patterns of the CDs (Mg–CDs, EDA–CDs, Mg–EDA–CDs,
Fig. S4†) showed a very broad peak as a matter of course.
Though the crystallization levels were related to carbonization
degree somewhat, the CDs without any doping exhibited almost
analogous particle size distribution and morphology (Fig. S5†)
compared with Mg–EDA–CDs. Thus, it could be inferred that
the great disparities in PL performances among these CDs
probably stem from their different surface states and
compositions.
It is commonly accepted that the uorescence emission
arises from the radiative recombination of the excitons trapped
17,25–27
by the defects on the surface of CDs.
So to investigate the
composition of CDs especially in the surface will help us to
understand the PL mechanism. It was revealed by FTIR results
(
1
Fig. S6†) that Mg–CDs showed more obvious n(C]O) peaks at
617 cm and n(C–O) peaks at 1076 cm compared with non-
ꢁ1
ꢁ1
doped CDs. While EDA–CDs exhibited n(N–H) peaks at
Fig. 2 XPS spectra of Mg–CDs (a), EDA–CDs (b) and Mg–EDA–CDs (c),
ꢁ1
ꢁ1
1555 cm
and n(C–N) peaks at 1391 cm
indicating the XPS C 1s spectra of Mg–CDs (d), EDA–CDs (e) and Mg–EDA–CDs (f).
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provided convincing evidence for the surface states and S12† when excited at different wavelengths, which means more
composition of the as-prepared CDs and the results were illus- choices for us to observe CDs labeled samples. Furthermore,
trated in Fig. 2. A 6.7% and 4.0% Mg atomic content were the CDs were observed mainly distributing over the cell
detected respectively from Mg–CDs and Mg–EDA–CDs, indi- membrane and the cytoplasmic area, while most cell nucleus
cating the successful introduction of Mg into the CDs in an showed very weak photoluminescence indicating there were
3
atomic state of Mg–CO with a same binding energy at around little CDs. This result was consistent with the previous reports
5
0 eV (shown in Fig. S7†). Moreover, their high-resolution C 1s that CDs were able to label both the cell membrane and the
9,16
spectra (Fig. 2d and f) showed strong signals at 288.6 eV cytoplasm but difficult to transport into the nucleus.
attributing to the oxygenated carbon atoms (C]O, O]C–O) Together with no blink reconrming the high photostability
compared with that of EDA–CDs, which means more carboxyl and the previous mentioned biocompatibility, Mg–EDA–CDs
groups xed on the CDs surface. As to EDA–CDs and Mg–EDA– proved their great potential in biomedical elds.
CDs, Fig. 2b and c reconrmed a 10.9% and 4.5% N atomic
content separately associated with N-doped surface passivation
by EDA.
Conclusion
19
The results obtained from above analyses demonstrate that In summary, we have explored a Mg/N double doping strategy to
the Mg–citric acid chelate in the carbon source plays dual roles fabricate highly luminescent CDs. The Mg–citric acid chelate in
in the hydrothermal process: introducing Mg and preserving the carbon source was utilized to introduce Mg and preserve
the carboxyl group by chelation. Meanwhile, EDA functions as more of carboxyl groups by chelation, which in conjunction
surface passivation reagent leading to doping N and amide–N with N passivation, contributed to a dramatic increase in the PL
contents. So, the Mg doping combined with N functionalization enhancement of the nal CDs. Our work demonstrated that the
contributed to the great PL enhancement of CDs. Importantly, resultant CDs are highly biocompatible and hold a great
this double doping acted in concert without mutual potential in biomedical applications.
interference.
The MTT assay method was used to evaluate the cytotoxicity
of as-prepared Mg–EDA–CDs before applications and the
Acknowledgements
results were demonstrated in Fig. S9.† The CDs exhibited low The authors gratefully acknowledge the support for this work
cytotoxicity with more than 90% cells retaining viability when from the National Natural Science Foundation of China (Grant
ꢁ
1
incubated in the medium containing 250 mg mL or lesser 51303210) and National Science and Technology Major Project
CDs. Thus an exploratory experiment was carried out to assess of China (Grant 2012ZX10004801-003).
the potential application of CDs for cell imaging. L929 cells
ꢁ
1
were cultured in the medium containing 100 mg mL CDs for
Notes and references
2
4 h, washed with PBS three times, xed with 4% para-
ꢀ
formaldehyde solution in PBS at 4 C overnight and then
observed under a laser scanning confocal microscope. It is
readily seen that CDs labeled L929 cells became bright excited
at 405 nm, 488 nm and 543 nm, respectively, whereas the
control cells (without CDs labeling, Fig. S10†) showed nearly
no visible uorescence detected under the same conditions.
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teristics (Fig. S11†) and uorescence microscopy photographs
of droplet containing CDs under bright eld, ultraviolet (330–
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