Phosphate-containing metabolites switch on phosphorescence of ferric ion engineered carbon dots in aqueous solution

Article abstract of DOI:10.1039/c4ra02592j

While most research has focused on the development of carbon dot (CD) based fluorescence sensors, much less attention has been paid to the phosphorescence phenomenon and its potential applications to date. Herein, room temperature phosphorescence (RTP) of water soluble CDs free of deoxidants and other inducers was observed for the first time in pure aqueous solution. RTP of CDs could be significantly quenched when chelating with iron ions as well as aggregation of CDs, presumably resulting from the formation of non-luminescent chelate. Due to a high affinity of iron ions to phosphate ions through well-known Fe-O-P bonds, the quenched RTP of functionalized CDs by Fe³⁺ could be basically recovered in the presence of phosphate-containing molecules. For a proof-of-concept demonstration, adenosine-5′-triphosphate (ATP), as a common phosphate-containing metabolite was quantitatively detected by a phosphorescence off-to-on approach. The enhancement of RTP at 440 nm was linearly proportional to the concentrations of ATP ranging from 20 to 200 μM with a detection limit as low as 14 μM. Moreover, the iron ion engineered CDs based RTP probe was used to estimate ATP levels in human blood plasma. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra02592j

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PAPER
Phosphate-containing metabolites switch on
phosphorescence of ferric ion engineered carbon
Cite this: RSC Adv., 2014, 4, 22318
dots in aqueous solution†
a
ab
ab
Meng-Xiang Su,ab Fang Yan,ab Bo Liab and Bin Di
*
*
Xiao Yan,‡ Jin-Long Chen,‡
While most research has focused on the development of carbon dot (CD) based fluorescence sensors,
much less attention has been paid to the phosphorescence phenomenon and its potential applications
to date. Herein, room temperature phosphorescence (RTP) of water soluble CDs free of deoxidants and
other inducers was observed for the first time in pure aqueous solution. RTP of CDs could be
significantly quenched when chelating with iron ions as well as aggregation of CDs, presumably resulting
from the formation of non-luminescent chelate. Due to a high affinity of iron ions to phosphate ions
through well-known Fe–O–P bonds, the quenched RTP of functionalized CDs by Fe3+ could be basically
recovered in the presence of phosphate-containing molecules. For a proof-of-concept demonstration,
adenosine-50-triphosphate (ATP), as a common phosphate-containing metabolite was quantitatively
detected by a phosphorescence “off-to-on” approach. The enhancement of RTP at 440 nm was linearly
proportional to the concentrations of ATP ranging from 20 to 200 mM with a detection limit as low as
14 mM. Moreover, the iron ion engineered CDs based RTP probe was used to estimate ATP levels in
human blood plasma.
Received 24th March 2014
Accepted 11th April 2014
DOI: 10.1039/c4ra02592j
www.rsc.org/advances
perturbation of a heavy atom, or on a solid support.12,5 However,
RTP emission from CDs dispersed in water has not been
Introduction
reported before. Interestingly, we have found that phosphores-
cence emissions can be observed in CDs dispersed in water at
room temperature.
Carbon dots (CDs) have attracted much attention in recent
years, owing to advantages such as excellent optical properties,
low toxicity, good biocompatibility, hydrophilicity and simple
synthesis.1–4 While most researches focus on the development
of CD based on uorescence, phosphorescence properties have
been paid much less attention. Room temperature phospho-
rescence (RTP) has been increasingly applied in sensing, bio-
imaging and anti-counterfeiting.5–7 On account of the emission
Phosphate-containing metabolites, which include nucleo-
side pyrophosphates, pyrophosphates, phosphates and phos-
phoproteins, play critical roles in biological processes.13 The
level of phosphate-containing metabolites can be used as an
indication of the orderly condition of biological systems. For
example, adenosine-50-triphosphate (ATP), an extracellular
signaling agent and the major energy carrier in cells, which has
not only been used as an indicator of cell viability, but also
associated with particular diseases such as angiocardiopathy
due to excessive production of ATP by creatine kinase.14–16 In
general, the physiological concentration of blood phosphate is
at a millimolar level, which mainly consists of ATP.17 Although
the traditional methods for determination of ATP levels in vitro
such as the luciferase assay, are sensitive, the enzyme and
substrates are costly and of uncertain stability.18 Considerable
efforts have been devoted towards designing a uorescence
probe to detect phosphorylated species, but the uorescence
might face interference due to the scattering of light or uo-
rescence from matrixes.19–21
delay, phosphorescence detection possesses
a signicant
advantage in that any uorescence emission and scattering
light could be stopped simply. In addition, only very few
compounds are able to emit analytically useful phosphores-
cence at room temperature.8 As is well known, RTP generally
originates from inorganics or metal complexes which might
represent a serious limitation due to known toxicity, potential
environment hazards and costliness.9–11 RTP emitted by CDs,
until now, has been observed only in assistant with the
aDepartment of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing
210009, China. E-mail: dibin@cpu.edu.cn; ddw888@vip.sina.com; Fax: +86
2583271269; Tel: +86 2593271269
bKey Laboratory of Drug Quality Control and Pharmacovigilance, China
Pharmaceutical University, Ministry of Education, Nanjing 210009, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra02592j
Herein, we demonstrate for the rst time a phosphorescence
“off-to-on” approach for sensing ATP by utilization of func-
tionalized CDs. The phosphorescence of CDs is signicantly
quenched when chelating with iron ions, then they are “turned-
‡ Jin-Long Chen and Xiao Yan contributed equally to this work.
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on” in the presence of phosphorylated species. Since ATP is the
main phosphate-containing metabolite in plasma, the proposed
sensing assay can be successfully used to rapidly estimate the
levels of phosphate-containing metabolites in plasma.
Determination of ATP in human blood plasma
Acetonitrile was rst added to an equal volume of human blood
plasma for protein precipitation. The mixture was centrifuged
at 5000 rpm for 5 minutes and the supernatant was collected.
ATP was spiked into the aliquots of supernatant and then the
mixture was diluted to a particular volume to make 10-fold
diluted human blood plasma that contains different concen-
trations of spiked ATP. Aliquots of ATP spiked 10-fold diluted
human blood plasma (500 mL) were then processed with the
aforementioned sensing procedure. Subsequently, the phos-
phorescence spectra were recorded.
Experimental section
Materials and apparatus
Citric acid, ethylenediamine, ferric trichloride hexahydrate
(FeCl3$6H2O), tris(hydroxymethyl)aminomethane were of
analytical grade and used without further purication. Adeno-
sine 50-triphosphate (ATP) disodium trihydrate was purchased
from Sigma-Aldrich and kept in the freezer under ꢀ20 ꢁC.
Human blood plasma was kindly provided by China Pharma-
ceutical University Hospital Department. All aqueous solutions
were prepared with deionized water puried through PL5242
Purelab Classic UV (PALL Co. Ltd., USA) to a resistivity of
18.2 MU cm.
Results and discussion
Morphological and structural characterizations of CDs
The CDs were prepared by use of a facile hydrothermal method.
The reaction was conducted by rst condensing citric acid and
ꢁ
ethylenediamine at 200 C for 5 hours, whereupon they formed
polymer-like CDs, which were then carbonized to form the CDs.
The morphology and structure of CDs were conrmed by the
Instrumentation
Phosphorescence measurements were performed on a Hitachi analysis described in the following. The transmission electron
F-4600 spectrouorometer (Hitachi Co. Ltd., Japan) in the microscopy (TEM) images (Fig. 1a and Fig. S1 in the ESI†) reveal
phosphorescence mode. Transmission electron microscopy the homogeneous CDs with spherical shape and almost uniform
(TEM) images were obtained from a TECNAI G2 F20 (FEI Co., diameter, about 11.5 nm. Fig. 1b also reveals the apparent
USA). UV-Vis absorption spectra were recorded with a Shimadzu aggregation of CDs induced by Fe3+ ions, suggesting that CDs
UV-2401 PC instrument (Shimadzu Co. Ltd., Japan). The FT-IR formed a chelate with Fe3+, thereby quenching the phosphores-
spectra (4000–400 cmꢀ1) in KBr were collected on a Jasco FT-IR- cence. The Atomic Force Microscope (AFM) images (Fig. S2 in the
4100 spectrometer (Jasco Co. Ltd., Japan). Time-resolved uo- ESI†) further indicates that the spherical CDs were well distrib-
rescence and phosphorescence decay by delay were performed uted. As seen from the FT-IR spectrum (Fig. S3 in the ESI†) that
on a FluoroMax-4 spectrouorometer (Horiba Jobin Yvon Co. stretching vibrations of C–OH at 3430 cmꢀ1, C–H at 2923 cmꢀ1
Ltd., France).
,
2850 cmꢀ1, 1476 cmꢀ1 and a broad peak at 1570 cmꢀ1 originate
from vibrations of aromatic C]C, asymmetric stretching vibra-
tions of C–NH–C at 1126 cmꢀ1, bending vibrations of N–H at 1570
cmꢀ1, and the vibrational absorption band of C]O at 1635 cmꢀ1
suggest those CDs produced by citric acid and ethylenediamine
containing C]C, C]O, –OH, –NH and –CH groups. Moreover,
1H-NMR spectroscopy was studied, in the 1H NMR spectrum
(Fig. S4 in the ESI†), the signals in the range of 6.5–8.5 ppm,
which correspond to aromatic ring hydrogen, were observed.
Preparation of phosphorescent CDs
Modifying from the previous work,22 0.42 g of citric acid and
535 mL of ethylenediamine were dissolved in 10 mL of deionized
water. Then the solution was transferred to a Teon-lined
stainless autoclave and heated at 200 ꢁC for 5 hours. The reactor
was cooled to room temperature, the resultant CDs solution was
puried by silica gel column with water as the developing
solvent. The puried CD solution was freeze-dried under
vacuum. Finally, the brown-black hygroscopic CDs were
obtained and stored in a dark desiccator at room temperature.
Phosphorescence properties of CDs
The CDs were produced from condensing citric acid and ethyl-
enediamine by use of a facile hydrothermal method under
Phosphorescence measurement
The “on-off-on” alternation of the phosphorescence of CDs was
performed in the Tris–HCl buffer at pH 8.0. To a 5.00 mL
volumetric ask, 1 mL of 10.0 mM Tris–HCl buffer, 250 mL of
1 mg mLꢀ1 aqueous CDs and 250 mL of 10.0 mM aqueous FeCl3
were sequentially added. Aer 5 min incubation at room
temperature, an appropriate volume of stock ATP solution was
added into the mixture to make the desired concentrations. The
mixture was diluted with deionized water to the volume, mixed
well and then incubated for 30 min at room temperature.
Subsequently, the phosphorescence intensity of the solution
was measured at lem/lex ¼ 440/360 nm.
Fig. 1 TEM images of (a) CDs alone, the inset shows the size distri-
bution of CDs and (b) CDs–Fe3+, apparent aggregation of CDs in white
dotted circle.
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different conditions in this work. As shown in Fig. 2 and 5d, the that CDs were equably dispersed in water over a large concen-
obtained CDs displayed a very strong photoluminescence tration range. Substantial phosphorescence integrated intensi-
including uorescence with a short lifetime of 14.4 ns and high ties with good signal-to-noise ratios and well-resolved emission
quantum yield to 75%, and phosphorescence with a long life- spectra were obtained with concentrations less than 1 mg of
time of 160 ms. Excitation/solvent-dependent emission behav- particles per litre. Furthermore, a series of CD samples were
iour (Fig. S5 in the ESI†) was observed, which is common in collected under different reaction temperatures and ratios of
uorescent carbon-containing nanoparticles related with the precursors. Results showed that reaction temperature gave a
surface state. In careful comparison to uorescence character- signicant effect on the phosphorescence intensity, while ratio
istics, we found that the phosphorescence emission prole of of precursors had a slight effect (Fig. S6 in the ESI†). It is worthy
CDs displayed only an enhancement in the longer wavelength to be noted that the photophysical characteristics described
region (500–600 nm) without the new emission band shied to above are different from those of common organic small
a long wavelength, similar to that of Mn doped ZnS quantum molecules. The corresponding clear-cut phosphorescence
dots.6 As demonstrated in the UV-Vis absorption spectrum mechanism needs to be further studied in the future. The above
(Fig. 2d), the peak centred at 240 nm is attributed to p–p* results of the synthetic CDs with high stability and high lumi-
transition of C]C, and the peak at 350 nm is attributed to the nescence quantum yield in aqueous solution were in favour of
n–p* transition of C]O. With the emission at 440 nm, the phosphorescence sensing and detection.
phosphorescence excitation spectrum shows a broad band from
300 to 400 nm and overlaps the band of C]O,23,24 suggesting
Effects of pH on the sensing of ATP
that the phosphorescence may come from the C]O bonds on
The effects of pH on the phosphorescence intensity of the CD
carbon dots. In addition, as for the aromatic carbonyl group, the
sensing system were investigated (Fig. 3). Phosphorescence
singlet and triplet states of the aromatic carbonyl group are
intensities of CDs only remain nearly constant over a wide range
close in energy, and the spin–orbit coupling is efficient, so that
of pH from 4.0 to 9.5 without changes in RTP emission prole.
it is prone to intersystem crossing.5 Therefore, it is reasonable to
The mixture solution of CDs and iron ions was observed with
suppose that the phosphorescence originates from the aromatic
the naked-eye. Precipitation occurred at a pH higher than 8.0
carbonyl group on CDs. Moreover, it is well known that poly-
cyclic aromatic hydrocarbons are a family of compounds which
due to hydrolysis, accompanied with the weakness of the
quenching capacity of Fe3+. However, for ATP sensing, a larger
can be directly determined by room temperature phosphorim-
phosphorescence recovery could be obtained at higher pH. In
etry.8 The phosphorescence of CDs is also possibly related to the
view of a comprehensive consideration of sensitivity and
graphitic structure which is similar to the polycyclic aromatic
reproducibility, a 10 mM Tris–HCl buffer of pH 8.0 was rec-
structure. Along with a serial of representative RTP emission
ommended to be used throughout.
spectra at different concentrations of CDs in water, no emission
wavelength shi was observed even at the lowest concentration.
A plot of the dependence of integrated RTP intensity versus
Selectivity of the CD-based phosphorescence method
nanoparticles concentration (or dilution factor) showing a good
linear relationship is shown in the inset in Fig. 2c. This shows
The considerable phosphorescence of the CDs was found to be
selectively quenched in the presence of Fe3+
. In addition, the
22
effects of various of other metal ions were investigated
including K+, Na+, Li+, Ca2+, Mg2+, Mn2+, Ba2+, Cu2+, Pb2+, Ag+,
Zn2+, Cd2+, Co2+, Ni2+ and Fe2+. As shown in Fig. 4a, only Fe3+
brought about the marked phosphorescence quenching. The
phosphorescence of CDs gradually decreased when the
concentration of Fe3+ ions increased. The quenching effect
presumably results from the effective electron transfer that
occurs from carboxyl and hydroxyl group functionalized CD
Fig. 2 (a) Phosphorescence spectra (solid line) and fluorescence
spectra (dash line) of CDs. (b) Time resolved phosphorescence decay Fig. 3 Effect of pH on the phosphorescence intensity of CDs (50 mg
by delay of CDs. (c) RTP emission profiles of different concentrations mLꢀ1) only, CDs (50 mg mLꢀ1) + Fe3+ (500 mM) and CDs (50 mg mLꢀ1) +
of CDs. (d) The UV-Vis absorption spectrum of CDs dispersed in water. Fe3+ (500 mM) + ATP (1200 mM), respectively.
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similar trend has also been observed in the time-resolved
uorescence decay prole. Besides, phosphorescence lifetime
of different concentration of CDs was shown in Fig. S10 (in the
ESI†), suggesting that the concentration of CDs has little impact
on the phosphorescence lifetime. These results described above
suggest that the phosphorescence quenching mechanism of
CDs by Fe3+ was attributed to a static quenching process
resulting from the formation of non-luminescent chelate, and
then the dissociation of Fe3+ from CDs by the phosphate species
through strong interactions.
Fig. 4 (a) Phosphorescence intensity of CDs (50 mg mLꢀ1) after
addition of different metal ions (500 mM). (b) Phosphorescence
enhancement of CDs (50 mg mLꢀ1)–Fe3+ (500 mM) system up addition
of different anions (100 mM). P and P0 are phosphorescence intensity
with and without anions, respectively.
Sensitivity of the sensing system
To demonstrate that the present system can be applied in
sensing of ATP, the phosphorescence responses to ATP at
different concentrations were measured under optimum
conditions. The phosphorescence emission spectra of the
system upon the addition of different concentrations of ATP
ranging from 20 to 1200 mM were shown in Fig. 6a. Fig. 6b
shows a dramatic increment of the phosphorescence intensity
as the concentration of ATP increased. Meanwhile, inset of
Fig. 6b clearly illustrates the corresponding calibration curve
plot between the phosphorescence intensity enhancement
factors (P ꢀ P0) against the ATP concentration. The linear
working concentration range was found to be 20–200 mM. The
regression equation is Y ¼ 0.113X + 1.398, with correlation
coefficient (R2) of 0.994. Moreover, the limit of detection (LOD)
was estimated to be 14 mM (3s/S), in which s is the standard
deviation for the blank solution (n ¼ 6), and S is the slope of the
calibration curve.
complexation with Fe3+. The quenching effect presumably
results from that the CDs and Fe3+ formed non-luminescent
chelate. Besides, it is widely known that phosphate ions give a
considerable affinity to iron ions through Fe–O–P bonds.21,25
Therefore, the quenching phosphorescence of CDs caused by
Fe3+ can be recovered in the presence of phosphate-containing
molecules. Hence, we further evaluated the selectivity of the
“off–on” phosphorescence approach to familiar anions such as
NO3ꢀ, SO42ꢀ, Acꢀ, HCO3ꢀ, formate, citrate, phenylalanine,
methionine, H2PO4ꢀ, HPO42ꢀ, PO43ꢀ, pyrophosphate and tri-
polyphosphate. As indicated in Fig. 4b, ATP has the greatest
effect on the restoration of the phosphorescence that is
quenched by Fe3+
.
Moreover, it is notable that multiphosphates show higher
affinity to Fe3+ than monophosphates (Fig. S8 in the ESI†).
Measuring ATP in real blood plasma samples
Incubation time and time-resolved phosphorescence decay
assays
In order to evaluate the applicability of the proposed CDs based
RTP “off-to-on” sensing approach, ATP assay in real human
blood plasma sample was further studied. As shown in Fig. 7a,
the uorescent background of blood plasma was signicant
To evaluate the effect of incubation time, the phosphorescence
intensity as a function of time was monitored. As illustrated in
Fig. S9 (in the ESI†), the chelation between CDs and Fe3+ was a
quite rapid process that reached equilibrium in 5 min. While
the formation and release of the ATP–Fe3+ complex from CDs
was relatively slow and achieved equilibrium in almost 30 min.
As seen from Fig. S7 (in the ESI†) that the linear relationship
between (P0 ꢀ P) and concentration of Fe3+ ions shows that the
phosphorescence quenching follows the Lineweaver–Burk
equation well, while the relationship between P0/P and the
concentration of Fe3+ does not follow Stern–Volmer's Equation.
According to references, the dynamic quenching can be
described by Stern–Volmer’s equation, while the static
quenching conforms to the Lineweaver–Burk equation.6 Thus, it
is reasonable to suppose that RTP of CDs quenched by Fe3+ ions
was in agreement with a static quenching model. To better
understand the RTP “off-to-on” mechanism for sensing ATP,
time-resolved phosphorescence and uorescence decay of CDs
in the presence of Fe3+ and ATP were studied, respectively.
Decay proles are shown in Fig. 5. The phosphorescence life-
time of free CDs is about 160 ms, while a slight increment of
lifetime of 166 ms was observed in the presence of Fe3+ ions, and
Fig. 5 (a) State phosphorescence spectra and time-resolved phos-
phorescence decay (b) of CDs alone, CDs + Fe3+, and CDs + Fe3+
+
ATP; (c) state fluorescence spectra and time-resolved fluorescence
the subsequent addition of ATP, with a lifetime of 159 ms. A decay (d) of CDs alone, CDs + Fe3+, and CDs + Fe3+ + ATP.
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coefficients (R2) of 0.991 and 0.993 respectively (Fig. 7c and d).
Using the standard addition method, we estimated that the
concentrations of ATP of the 2 human blood plasma samples
are 6.77 mM and 4.07 mM respectively. As previously
mentioned, the physiological concentration of blood phos-
phates is at a millimolar level, and is mainly dominated by ATP.
Thus we considered that ATP was mainly responsible for the
phosphorescence recovery in the sensing process. Conse-
quently, the proposed sensing system can be used to evaluate
the health conditions of individuals.
Fig. 6 (a) Phosphorescence emission spectra of CDs–Fe3+ (50 mg
mLꢀ1–500 mM) in the presence of different concentrations of ATP (0,
20, 50, 100, 150, 200, 500, 800, 1200 mM). (b) The relationship
between (P ꢀ P0) and the concentration of ATP. P and P0 are phos-
phorescence intensity with and without ATP, respectively.
Conclusions
In summary, we have demonstrated a novel RTP method based
on the phosphorescence property of CDs for the cost-effective,
readily, sensitive detection of ATP, the main phosphate-con-
taining metabolite in human blood plasma. The phosphores-
cence emission centred at 440 nm with an average lifetime of
160 ms under excitation of 360 nm can be quenched in the
presence of Fe3+ attributing to nonradiative electron transfer
and then turned on by the phosphate ions through the strong
interactions. Deoxidants and other inducers that are necessary
in conventional RTP detection and interferences from auto-
uorescence and the scattering light of the complex matrix
encountered in spectrouorometry could be readily avoided in
the presence of CD-based RTP. Combinations of varieties of
interactions between metal ions and surface ligands of CDs and
the desirable phosphorescence properties show that water-
soluble and biocompatible CDs are promising for RTP chemical
and biological sensing and time-resolved imaging. Moreover,
CD-based RTP nanocomposites could potentially be used in
anti-counterfeiting, especially for food and drugs.
(black line), while no background was observed in the phos-
phorescence mode (red line). Since only very few species are
able to emit analytically useful phosphorescence at room
temperature,8 the long delay time of phosphorescence can
simply avoid the interference from the scattering light and
undesired uorescence emission.
A standard addition method was applied to estimate the ATP
concentration in human blood plasma. Different concentra-
tions of ATP were spiked into the 10-fold diluted human blood
plasma and were further incubated with the CDs–Fe3+ sensing
system under the optimal conditions. In the test solution,
human blood plasma was nally diluted 100-fold and the
spiked ATP was at a concentration of 0, 20, 50, 100, 150 mM. The
blood plasma samples from
2 healthy volunteers were
measured and the results are shown in Fig. 7b. It was found that
the present approach provides a linear response to ATP spiked
into human blood plasma, and the regression equations are
Y ¼ 0.102X + 6.903 and Y ¼ 0.083X + 3.422, with correlation
Acknowledgements
The authors gratefully acknowledge the nancial support for
this work from the National Natural Science Foundation of
China (Grant no. 21305161), the Natural Science Foundation of
Jiangsu Province (Grant no. BK20130643).
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